Negatively charged peg-lipid conjugates

ABSTRACT

The invention provides PEG-conjugated lipids of formula (I): A-B-C (I) wherein A, B, and C have any of the values defined in the specification, as well as compositions comprising the PEG-conjugated lipids of formula (I), nucleic acid lipid nanoparticles comprising the PEG-conjugated lipids of formula (I), and methods of using the PEG-conjugated lipids of formula (I), the compositions, and the nucleic acid nanoparticles.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This patent application claims the benefit of priority of U.S.application Ser. No. 62/758,099, filed Nov. 9, 2018, which applicationis herein incorporated by reference.

BACKGROUND

Nanoparticles comprising PEG-lipids have been used for the delivery of avariety of active agents and therapeutic agents. Typically, PEG-lipidsare prepared by derivatization of the polar head group of adiacylglycerophospholipid such as distearoylphosphatidylethanolamine(DSPE), with PEG. These phospholipids usually contain two fatty acylchains bonded to the 1- and 2-position of glycerol by ester linkages.Unfortunately, these acyl groups are susceptible to cleavage underacidic or basic conditions. The resulting hydrolytic products, such asanalogs of lysophospholipid and glycerophosphate, do not remainassociated with the bilayer structure of the lipid particle.Unfortunately, such dissociation can weaken the integrity of the lipidparticle, leading to significant leakage of the bioactive agent or drugfrom the lipid particle and contributing to instability during storage,and thus shortened shelf-life of the lipid particle product. Inaddition, the loss of these hydrolysis products, such asPEG-lysophospholipid, from the lipid particle negates the benefitsotherwise resulting from the presence of the PEG-phospholipid.

Currently there is a need for additional PEG-conjugated lipids havingvaried physical and chemical properties. In particular there is a needfor PEG-conjugated lipids for incorporation into lipid-nucleic acidparticles (LNPs) that are useful for delivering mRNA. There is a needfor PEG-conjugated lipids for incorporation into LNPs that are usefulfor delivering nucleic acids by intramuscular injection. The presentinvention addresses this and other needs.

SUMMARY

The invention provides novel, PEG-conjugated lipids that comprise one ormore anionic precursor groups. These novel PEG-conjugated lipids areparticularly useful as components of LNPs that are useful for deliveringnucleic acids, such as mRNA. They are also particularly useful ascomponents of LNPs that are formulated for intramuscular injection.

In one embodiment the invention provides a PEG-conjugated lipid offormula (I):

A-B-C  (I)

wherein:

A is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, or (C₂-C₆)alkanoyloxy, whereinany (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, and (C₂-C₆)alkanoyloxy issubstituted with one or more anionic precursor groups, and wherein any(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, and (C₂-C₆)alkanoyloxy isoptionally substituted with one or more groups independently selectedfrom the group consisting of halo, hydroxyl, (C₁-C₃)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₁-C₃)alkylthio, or(C₂-C₃)alkanoyloxy;

B is a polyethylene glycol chain having a molecular weight of from about550 daltons to about 10,000 daltons;

C is -L-R^(a)

L is selected from the group consisting of a direct bond, —C(O)O—,—C(O)NR^(b)—, —NR^(b)—, —C(O)—, —NR^(b)C(O)O—, —NR^(b)C(O)NR^(b)—,—S—S—, —O—, —(O)CCH₂CH₂C(O)—, and —NHC(O)CH₂CH₂C(O)NH—;

R^(a) is a branched (C₁₀-C₅₀)alkyl or branched (C₁₀-C₅₀)alkenyl whereinone or more carbon atoms of the branched (C₁₀-C₅₀)alkyl or branched(C₁₀-C₅₀)alkenyl have been replaced with —O—; and each R^(b) isindependently H or (C₁-C₆)alkyl.

In yet another aspect, the invention provides, a nucleic acid-lipidparticle comprising: one or more nucleic acid molecules; a cationiclipid; a non-cationic lipid; and a compound of formula (I); wherein theone or more nucleic acid molecules are encapsulated within the lipidparticle.

In yet another aspect, the invention provides, a pharmaceuticalcomposition comprising a nucleic acid-lipid particle of any one ofclaims 45-74, and a pharmaceutically acceptable carrier.

In yet another aspect, the invention provides, a composition comprising,about 1.5% of total lipid of the compound:

about 50.0% of total lipid of the compound:

about 38.5% of total lipid of cholesterol; and about 10.0% of totallipid of DSPC.

In yet another aspect, the invention provides, a composition comprisingabout 2.0% of total lipid of the compound:

about 40.0% of total lipid of the compound:

about 48.5% of total lipid of cholesterol; and about 10.0% of totallipid of DSPC.

In yet another aspect, the invention provides, a composition comprising,about 1.6% of total lipid of the compound:

about 54.9% of total lipid of the compound:

about 32.8% of total lipid of cholesterol; and about 10.0% of totallipid of DSPC.

In yet another aspect, the invention provides, a method of introducing anucleic acid into a cell, comprising contacting said cell with a nucleicacid-lipid particle of the invention or a composition of the invention.

In yet another aspect, the invention provides, a nucleic acid-lipidparticle of the invention or a composition of the invention for use inthe in vivo delivery of a nucleic acid to a mammal.

In yet another aspect, the invention provides, the use of a nucleicacid-lipid particle of the invention or a composition of the inventionto prepare a medicament for the in vivo delivery of a nucleic acid to amammal.

In yet another aspect, the invention provides, a method for treating adisease or disorder in a mammalian subject in need thereof, the methodcomprising: administering to the mammalian subject a therapeuticallyeffective amount of nucleic acid-lipid particle of the invention.

In yet another aspect, the invention provides, a nucleic acid-lipidparticle of the invention or a composition of the invention for use intreating a disease or disorder in a mammal.

In yet another aspect, the invention provides, the use of a nucleicacid-lipid particle of the invention or a composition of the inventionto prepare a medicament for treating a disease or disorder in a mammal.

The invention also provides processes and intermediates disclosed hereinthat are useful for making the compounds, formulations, or nanoparticlesof the invention.

Other features, objects and advantages of the invention and itspreferred embodiments will become apparent from the detaileddescription, examples, claims and figures that follow.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “anionic precursor group” includes groups that are capable offorming an ion at physiological pH. For example, the term includes thegroups —CO₂H, —O—P(═O)(OH)₂, —OS(═O)₂(OH), —O—S(═O)(OH), and —B(OH)₂. Inone embodiment, the anionic precursor is —CO₂H.

The term “cycloalkyl” refers to a saturated or partially unsaturated(non-aromatic) all carbon ring having 3 to 8 carbon atoms. Non-limitingexamples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl,and cyclohexyl.

The term “alkyl”, by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain hydrocarbonradical, having the number of carbon atoms designated (i.e., C₁₋₈ meansone to eight carbons). Examples include (C₁-C₈)alkyl, (C₂-C₈)alkyl,C₁-C₆)alkyl, (C₂-C₆)alkyl and (C₃-C₆)alkyl. Examples of alkyl groupsinclude methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl,iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and andhigher homologs and isomers.

The term “alkenyl” refers to an unsaturated alkyl radical having one ormore double bonds. Examples of such unsaturated alkyl groups includevinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl) and the higher homologs andisomers.

The term “alkynyl” refers to an unsaturated alkyl radical having one ormore triple bonds. An alkynyl group may also have one or more doublebonds in addition to the one or more triple bonds. Examples of suchunsaturated alkyl groups ethynyl, 1- and 3-propynyl, 3-butynyl,2-hexene-5-ynyl, and higher homologs and isomers.

The term “interfering RNA” or “RNAi” or “interfering RNA sequence”refers to single-stranded RNA (e.g., mature miRNA) or double-strandedRNA (i.e., duplex RNA such as siRNA, aiRNA, or pre-miRNA) that iscapable of reducing or inhibiting the expression of a target gene orsequence (e.g., by mediating the degradation or inhibiting thetranslation of mRNAs which are complementary to the interfering RNAsequence) when the interfering RNA is in the same cell as the targetgene or sequence. Interfering RNA thus refers to the single-stranded RNAthat is complementary to a target mRNA sequence or to thedouble-stranded RNA formed by two complementary strands or by a single,self-complementary strand. Interfering RNA may have substantial orcomplete identity to the target gene or sequence, or may comprise aregion of mismatch (i.e., a mismatch motif). The sequence of theinterfering RNA can correspond to the full-length target gene, or asubsequence thereof.

Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g.,interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides inlength, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotidesin length, for example, about 20-24, 21-22, or 21-23 (duplex)nucleotides in length (e.g., each complementary sequence of thedouble-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25nucleotides in length, for example, about 20-24, 21-22, or 21-23nucleotides in length, and the double-stranded siRNA is about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, for example,about 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes maycomprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 toabout 3 nucleotides and 5′ phosphate termini. Examples of siRNA include,without limitation, a double-stranded polynucleotide molecule assembledfrom two separate stranded molecules, wherein one strand is the sensestrand and the other is the complementary antisense strand; adouble-stranded polynucleotide molecule assembled from a single strandedmolecule, where the sense and antisense regions are linked by a nucleicacid-based or non-nucleic acid-based linker; a double-strandedpolynucleotide molecule with a hairpin secondary structure havingself-complementary sense and antisense regions; and a circularsingle-stranded polynucleotide molecule with two or more loop structuresand a stem having self-complementary sense and antisense regions, wherethe circular polynucleotide can be processed in vivo or in vitro togenerate an active double-stranded siRNA molecule.

Typically, siRNA are chemically synthesized. siRNA can also be generatedby cleavage of longer dsRNA (e.g., dsRNA greater than about 25nucleotides in length) with the E. coli RNase III or Dicer. Theseenzymes process the dsRNA into biologically active siRNA (see, e.g.,Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegariet al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al.,Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res.,31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); andRobertson et al., J. Biol. Chem., 243:82 (1968)). Typically, dsRNA areat least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotidesin length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotidesin length, or longer. The dsRNA can encode for an entire gene transcriptor a partial gene transcript. In certain instances, siRNA may be encodedby a plasmid (e.g., transcribed as sequences that automatically foldinto duplexes with hairpin loops).

As used herein, the term “mismatch motif” or “mismatch region” refers toa portion of an interfering RNA (e.g., siRNA, aiRNA, miRNA) sequencethat does not have 100% complementarity to its target sequence. Aninterfering RNA may have at least one, two, three, four, five, six, ormore mismatch regions. The mismatch regions may be contiguous or may beseparated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.The mismatch motifs or regions may comprise a single nucleotide or maycomprise two, three, four, five, or more nucleotides.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a nucleic acid (e.g., an interferingRNA or mRNA) is an amount sufficient to produce the desired effect,e.g., an inhibition of expression of a target sequence in comparison tothe normal expression level detected in the absence of an interferingRNA; or mRNA-directed expression of an amount of a protein that causes adesirable biological effect in the organism within which the protein isexpressed. Inhibition of expression of a target gene or target sequenceis achieved when the value obtained with an interfering RNA relative tothe control is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other embodiments, theexpressed protein is an active form of a protein that is normallyexpressed in a cell type within the body, and the therapeuticallyeffective amount of the mRNA is an amount that produces an amount of theencoded protein that is at least 50% (e.g., at least 60%, or at least70%, or at least 80%, or at least 90%) of the amount of the protein thatis normally expressed in the cell type of a healthy individual. Suitableassays for measuring expression of a target gene or target sequenceinclude, e.g., examination of protein or RNA levels using techniquesknown to those of skill in the art such as dot blots, northern blots, insitu hybridization, ELISA, immunoprecipitation, enzyme function, as wellas phenotypic assays known to those of skill in the art.

By “decrease,” “decreasing,” “reduce,” or “reducing” of an immuneresponse by an interfering RNA is intended to mean a detectable decreaseof an immune response to a given interfering RNA (e.g., a modifiedinterfering RNA). The amount of decrease of an immune response by amodified interfering RNA may be determined relative to the level of animmune response in the presence of an unmodified interfering RNA. Adetectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or morelower than the immune response detected in the presence of theunmodified interfering RNA. A decrease in the immune response tointerfering RNA is typically measured by a decrease in cytokineproduction (e.g., IFNγ, IFNα, TNFα, IL-6, or IL-12) by a responder cellin vitro or a decrease in cytokine production in the sera of a mammaliansubject after administration of the interfering RNA.

By “decrease,” “decreasing,” “reduce,” or “reducing” of an immuneresponse by an mRNA is intended to mean a detectable decrease of animmune response to a given mRNA (e.g., a modified mRNA). The amount ofdecrease of an immune response by a modified mRNA may be determinedrelative to the level of an immune response in the presence of anunmodified mRNA. A detectable decrease can be about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, or more lower than the immune response detected in thepresence of the unmodified mRNA. A decrease in the immune response tomRNA is typically measured by a decrease in cytokine production (e.g.,IFNγ, IFNα, TNFα, IL-6, or IL-12) by a responder cell in vitro or adecrease in cytokine production in the sera of a mammalian subject afteradministration of the mRNA.

As used herein, the term “responder cell” refers to a cell, typically amammalian cell, which produces a detectable immune response whencontacted with an immunostimulatory interfering RNA such as anunmodified siRNA. Exemplary responder cells include, e.g., dendriticcells, macrophages, peripheral blood mononuclear cells (PBMCs),splenocytes, and the like. Detectable immune responses include, e.g.,production of cytokines or growth factors such as TNF-α, IFN-α, IFN-β,IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TGF, andcombinations thereof.

“Substantial identity” refers to a sequence that hybridizes to areference sequence under stringent conditions, or to a sequence that hasa specified percent identity over a specified region of a referencesequence.

The phrase “stringent hybridization conditions” refers to conditionsunder which a nucleic acid will hybridize to its target sequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, for example, 10 times background hybridization.

Exemplary stringent hybridization conditions can be as follows: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec.-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y. (1990).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous references, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds.

The terms “substantially identical” or “substantial identity,” in thecontext of two or more nucleic acids, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same (i.e., at least about 60%, typically atleast about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over aspecified region), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. This definition, when the context indicates, alsorefers analogously to the complement of a sequence. Typically, thesubstantial identity exists over a region that is at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of a number of contiguous positions selected from the groupconsisting of from about 5 to about 60, usually about 10 to about 45,more usually about 15 to about 30, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.,48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology, Ausubelet al., eds. (1995 supplement)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.,25:3389-3402 (1977) and Altschul et al., J. Mol. Biol., 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids of the invention. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/).

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, for example, less than about 0.01or less than about 0.001.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA and RNA. DNA may be in the formof, e.g., antisense molecules, plasmid DNA, precondensed DNA, a PCRproduct, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expressioncassettes, chimeric sequences, chromosomal DNA, or derivatives andcombinations of these groups. RNA may be in the form of siRNA,asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA,rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, and combinationsthereof. Nucleic acids include nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid.

Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, SNPs, and complementary sequences aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups. “Bases” include purines and pyrimidines, which furtherinclude natural compounds adenine, thymine, guanine, cytosine, uracil,inosine, and natural analogs, and synthetic derivatives of purines andpyrimidines, which include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids; and (3) “derived lipids” such assteroids.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids; and (3) “derived lipids” such assteroids.

As used herein, the term “LNP” refers to a lipid-nucleic acid particleor a nucleic acid-lipid particle (e.g., a stable nucleic acid-lipidparticle). A LNP represents a particle made from lipids (e.g., acationic lipid, a non-cationic lipid, and a conjugated lipid thatprevents aggregation of the particle), and a nucleic acid, wherein thenucleic acid (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, shorthairpin RNA (shRNA), dsRNA, mRNA, self-amplifying RNA, or a plasmid,including plasmids from which an interfering RNA or mRNA is transcribed)is encapsulated within the lipid. In one embodiment, the nucleic acid isat least 50% encapsulated in the lipid; in one embodiment, the nucleicacid is at least 75% encapsulated in the lipid; in one embodiment, thenucleic acid is at least 90% encapsulated in the lipid; and in oneembodiment, the nucleic acid is completely encapsulated in the lipid.LNPs typically contain a cationic lipid, a non-cationic lipid, and alipid conjugate (e.g., a PEG-lipid conjugate). LNP are extremely usefulfor systemic applications, as they can exhibit extended circulationlifetimes following intravenous (i.v.) injection, they can accumulate atdistal sites (e.g., sites physically separated from the administrationsite), and they can mediate expression of the transfected gene orsilencing of target gene expression at these distal sites.

The lipid particles of the invention (e.g., LNPs) typically have a meandiameter of from about 40 nm to about 150 nm, from about 50 nm to about150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110nm, or from about 70 to about 90 nm, and are substantially non-toxic. Inaddition, nucleic acids, when present in the lipid particles of theinvention, are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and20070042031, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

As used herein, “lipid encapsulated” can refer to a lipid particle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA or mRNA), with full encapsulation, partialencapsulation, or both. In one embodiment, the nucleic acid is fullyencapsulated in the lipid particle (e.g., to form an LNP, or othernucleic acid-lipid particle).

The term “lipid conjugate” refers to a conjugated lipid that inhibitsaggregation of lipid particles. Such lipid conjugates include, but arenot limited to, polyamide oligomers (e.g., ATTA-lipid conjugates),PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEGcoupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled tophosphatidylethanolamines, PEG conjugated to ceramides (see, e.g., U.S.Pat. No. 5,885,613, the disclosure of which is herein incorporated byreference in its entirety for all purposes), cationic PEG lipids, andmixtures thereof. PEG can be conjugated directly to the lipid or may belinked to the lipid via a linker moiety. Any linker moiety suitable forcoupling the PEG to a lipid can be used including, e.g., non-estercontaining linker moieties and ester-containing linker moieties. In someembodiments, non-ester containing linker moieties are used.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long-chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic, or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,amino lipids, and sphingolipids.

Representative examples of phospholipids include, but are not limitedto, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, are also within the group designated as amphipathiclipids. Additionally, the amphipathic lipids described above can bemixed with other lipids including triglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “cationic lipid” refers to any of a number of lipid speciesthat carry a net positive charge at a selected pH, such as physiologicalpH (e.g., pH of about 7.0). It has been surprisingly found that cationiclipids comprising alkyl chains with multiple sites of unsaturation,e.g., at least two or three sites of unsaturation, are particularlyuseful for forming lipid particles with increased membrane fluidity. Anumber of cationic lipids and related analogs, which are also useful inthe present invention, have been described in U.S. Patent PublicationNos. 20060083780 and 20060240554; U.S. Pat. Nos. 5,208,036; 5,264,618;5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No.WO 96/10390, the disclosures of which are herein incorporated byreference in their entirety for all purposes. Non-limiting examples ofcationic lipids are described in detail herein. In some cases, thecationic lipids comprise a protonatable tertiary amine (e.g., pHtitratable) head group, C18 alkyl chains, ether linkages between thehead group and alkyl chains, and 0 to 3 double bonds. Such lipidsinclude, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The term “fusogenic” refers to the ability of a lipid particle, such asa LNP, to fuse with the membranes of a cell. The membranes can be eitherthe plasma membrane or membranes surrounding organelles, e.g., endosome,nucleus, etc.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

“Distal site,” as used herein, refers to a physically separated site,which is not limited to an adjacent capillary bed, but includes sitesbroadly distributed throughout an organism.

“Serum-stable” in relation to nucleic acid-lipid particles such as LNPmeans that the particle is not significantly degraded after exposure toa serum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of lipidparticles that leads to a broad biodistribution of an active agent ortherapeutic agent, such as an interfering RNA or mRNA, within anorganism. Some techniques of administration can lead to the systemicdelivery of certain agents, but not others. Systemic delivery means thata useful, typically therapeutic, amount of an agent is exposed to mostparts of the body. To obtain broad biodistribution generally requires ablood lifetime such that the agent is not rapidly degraded or cleared(such as by first pass organs (liver, lung, etc.) or by rapid,nonspecific cell binding) before reaching a disease site distal to thesite of administration. Systemic delivery of lipid particles can be byany means known in the art including, for example, intravenous,subcutaneous, and intraperitoneal. In one embodiment, systemic deliveryof lipid particles is by intravenous delivery.

“Local delivery,” as used herein, refers to delivery of an active agentor therapeutic agent, such as an interfering RNA or mRNA, directly to atarget site within an organism. For example, an agent can be locallydelivered by direct injection into a disease site such as a tumor orother target site such as a site of inflammation or a target organ suchas the liver, heart, pancreas, kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

The term “cancer” refers to any member of a class of diseasescharacterized by the uncontrolled growth of aberrant cells. The termincludes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer,small intestine cancer, stomach (gastric) cancer, esophageal cancer;gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer,breast cancer, ovarian cancer; cervical cancer, prostate cancer, renalcancer (e.g., renal cell carcinoma), cancer of the central nervoussystem, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head andneck cancers, osteogenic sarcomas, and blood cancers. Non-limitingexamples of specific types of liver cancer include hepatocellularcarcinoma (HCC), secondary liver cancer (e.g., caused by metastasis ofsome other non-liver cancer cell type), and hepatoblastoma. As usedherein, a “tumor” comprises one or more cancerous cells.

I. DESCRIPTION OF THE EMBODIMENTS

The present invention provides novel, serum-stable lipid particlescomprising one or more active agents or therapeutic agents, methods ofmaking the lipid particles, and methods of delivering and/oradministering the lipid particles (e.g., for the treatment of a diseaseor disorder).

In one aspect, the present invention provides lipid particlescomprising: (a) one or more active agents or therapeutic agents; (b) oneor more cationic lipids comprising from about 40 mol % to about 85 mol %of the total lipid present in the particle; (c) one or more non-cationiclipids comprising from about 13 mol % to about 49.5 mol % of the totallipid present in the particle; and (d) one or more PEG-conjugated lipidsof formula (I) comprising from about 0.1 mol % to about 10% mol % of thetotal lipid present in the particle.

In one aspect, the present invention provides lipid particlescomprising: (a) one or more active agents or therapeutic agents; (b) oneor more cationic lipids comprising from about 40 mol % to about 85 mol %of the total lipid present in the particle; (c) one or more non-cationiclipids comprising from about 13 mol % to about 49.5 mol % of the totallipid present in the particle; and (d) one or more PEG-conjugated lipidsof formula (I) comprising from about 0.5 mol % to about 2 mol % of thetotal lipid present in the particle.

In one aspect, the present invention provides lipid particlescomprising: (a) one or more active agents or therapeutic agents; (b) oneor more cationic lipids comprising from about 50 mol % to about 85 mol %of the total lipid present in the particle; (c) one or more non-cationiclipids comprising from about 13 mol % to about 49.5 mol % of the totallipid present in the particle; and (d) one or more PEG-conjugated lipidsof formula (I) comprising from about 0.1 mol % to about 10% mol % of thetotal lipid present in the particle.

In one aspect, the present invention provides lipid particlescomprising: (a) one or more active agents or therapeutic agents; (b) oneor more cationic lipids comprising from about 50 mol % to about 85 mol %of the total lipid present in the particle; (c) one or more non-cationiclipids comprising from about 13 mol % to about 49.5 mol % of the totallipid present in the particle; and (d) one or more PEG-conjugated lipidsof formula (I) comprising from about 0.5 mol % to about 2 mol % of thetotal lipid present in the particle.

In certain embodiments, the active agent or therapeutic agent is fullyencapsulated within the lipid portion of the lipid particle such thatthe active agent or therapeutic agent in the lipid particle is resistantin aqueous solution to enzymatic degradation, e.g., by a nuclease orprotease. In certain other embodiments, the lipid particles aresubstantially non-toxic to mammals such as humans.

In some embodiments, the active agent or therapeutic agent comprises anucleic acid. In certain instances, the nucleic acid comprises aninterfering RNA molecule such as, e.g., an siRNA, aiRNA, miRNA, ormixtures thereof. In certain other instances, the nucleic acid comprisessingle-stranded or double-stranded DNA, RNA, or a DNA/RNA hybrid suchas, e.g., an antisense oligonucleotide, a ribozyme, a plasmid, animmunostimulatory oligonucleotide, or mixtures thereof. In certain otherinstances, the nucleic acid comprises one or more mRNA molecules (e.g.,a cocktail).

In other embodiments, the active agent or therapeutic agent comprises apeptide or polypeptide. In certain instances, the peptide or polypeptidecomprises an antibody such as, e.g., a polyclonal antibody, a monoclonalantibody, an antibody fragment; a humanized antibody, a recombinantantibody, a recombinant human antibody, a Primatized™ antibody, ormixtures thereof. In certain other instances, the peptide or polypeptidecomprises a cytokine, a growth factor, an apoptotic factor, adifferentiation-inducing factor, a cell-surface receptor, a ligand, ahormone, a small molecule (e.g., small organic molecule or compound), ormixtures thereof.

In one embodiments, the active agent or therapeutic agent comprises ansiRNA. In one embodiment, the siRNA molecule comprises a double-strandedregion of about 15 to about 60 nucleotides in length (e.g., about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, or 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The siRNAmolecules of the invention are capable of silencing the expression of atarget sequence in vitro and/or in vivo.

In some embodiments, the siRNA molecule comprises at least one modifiednucleotide. In certain embodiments, the siRNA molecule comprises one,two, three, four, five, six, seven, eight, nine, ten, or more modifiednucleotides in the double-stranded region. In certain instances, thesiRNA comprises from about 1% to about 100% (e.g., about 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%) modified nucleotides in the double-strandedregion. In some embodiments, less than about 25% (e.g., less than about25%, 20%, 15%, 10%, or 5%) or from about 1% to about 25% (e.g., fromabout 1%-25%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, or 10%-20%) of thenucleotides in the double-stranded region comprise modified nucleotides.

In other embodiments, the siRNA molecule comprises modified nucleotidesincluding, but not limited to, 2′-O-methyl (2′OMe) nucleotides,2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, and mixtures thereof. In some embodiments, the siRNAcomprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosinenucleotides, and mixtures thereof. In certain instances, the siRNA doesnot comprise 2′OMe-cytosine nucleotides. In other embodiments, the siRNAcomprises a hairpin loop structure.

The siRNA may comprise modified nucleotides in one strand (i.e., senseor antisense) or both strands of the double-stranded region of the siRNAmolecule. Typically, uridine and/or guanosine nucleotides are modifiedat selective positions in the double-stranded region of the siRNAduplex. With regard to uridine nucleotide modifications, at least one,two, three, four, five, six, or more of the uridine nucleotides in thesense and/or antisense strand can be a modified uridine nucleotide suchas a 2′OMe-uridine nucleotide. In some embodiments, every uridinenucleotide in the sense and/or antisense strand is a 2′OMe-uridinenucleotide. With regard to guanosine nucleotide modifications, at leastone, two, three, four, five, six, or more of the guanosine nucleotidesin the sense and/or antisense strand can be a modified guanosinenucleotide such as a 2′OMe-guanosine nucleotide. In some embodiments,every guanosine nucleotide in the sense and/or antisense strand is a2′OMe-guanosine nucleotide.

In certain embodiments, at least one, two, three, four, five, six,seven, or more 5′-GU-3′ motifs in an siRNA sequence may be modified,e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/orby introducing modified nucleotides such as 2′OMe nucleotides. The5′-GU-3′ motif can be in the sense strand, the antisense strand, or bothstrands of the siRNA sequence. The 5′-GU-3′ motifs may be adjacent toeach other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more nucleotides.

In some embodiments, a modified siRNA molecule is less immunostimulatorythan a corresponding unmodified siRNA sequence. In such embodiments, themodified siRNA molecule with reduced immunostimulatory propertiesadvantageously retains RNAi activity against the target sequence. Inanother embodiment, the immunostimulatory properties of the modifiedsiRNA molecule and its ability to silence target gene expression can bebalanced or optimized by the introduction of minimal and selective 2′OMemodifications within the siRNA sequence such as, e.g., within thedouble-stranded region of the siRNA duplex. In certain instances, themodified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that the immunostimulatory properties of the modifiedsiRNA molecule and the corresponding unmodified siRNA molecule can bedetermined by, for example, measuring INF-α and/or IL-6 levels fromabout two to about twelve hours after systemic administration in amammal or transfection of a mammalian responder cell using anappropriate lipid-based delivery system (such as the LNP delivery systemdisclosed herein).

In certain embodiments, a modified siRNA molecule has an IC₅₀ (i.e.,half-maximal inhibitory concentration) less than or equal to ten-foldthat of the corresponding unmodified siRNA (i.e., the modified siRNA hasan IC₅₀ that is less than or equal to ten-times the IC₅₀ of thecorresponding unmodified siRNA). In other embodiments, the modifiedsiRNA has an IC₅₀ less than or equal to three-fold that of thecorresponding unmodified siRNA sequence. In yet other embodiments, themodified siRNA has an IC₅₀ less than or equal to two-fold that of thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that a dose-response curve can be generated and theIC₅₀ values for the modified siRNA and the corresponding unmodifiedsiRNA can be readily determined using methods known to those of skill inthe art.

In yet another embodiment, a modified siRNA molecule is capable ofsilencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of theexpression of the target sequence relative to the correspondingunmodified siRNA sequence.

In some embodiments, the siRNA molecule does not comprise phosphatebackbone modifications, e.g., in the sense and/or antisense strand ofthe double-stranded region. In other embodiments, the siRNA comprisesone, two, three, four, or more phosphate backbone modifications, e.g.,in the sense and/or antisense strand of the double-stranded region. Insome embodiments, the siRNA does not comprise phosphate backbonemodifications.

In further embodiments, the siRNA does not comprise 2′-deoxynucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region. In yet further embodiments, the siRNA comprisesone, two, three, four, or more 2′-deoxy nucleotides, e.g., in the senseand/or antisense strand of the double-stranded region. In someembodiments, the siRNA does not comprise 2′-deoxy nucleotides.

In certain instances, the nucleotide at the 3′-end of thedouble-stranded region in the sense and/or antisense strand is not amodified nucleotide. In certain other instances, the nucleotides nearthe 3′-end (e.g., within one, two, three, or four nucleotides of the3′-end) of the double-stranded region in the sense and/or antisensestrand are not modified nucleotides.

The siRNA molecules described herein may have 3′ overhangs of one, two,three, four, or more nucleotides on one or both sides of thedouble-stranded region, or may lack overhangs (i.e., have blunt ends) onone or both sides of the double-stranded region. Typically, the siRNAhas 3′ overhangs of two nucleotides on each side of the double-strandedregion. In certain instances, the 3′ overhang on the antisense strandhas complementarity to the target sequence and the 3′ overhang on thesense strand has complementarity to a complementary strand of the targetsequence. Alternatively, the 3′ overhangs do not have complementarity tothe target sequence or the complementary strand thereof. In someembodiments, the 3′ overhangs comprise one, two, three, four, or morenucleotides such as 2′-deoxy (2′H) nucleotides. In certain embodiments,the 3′ overhangs comprise deoxythymidine (dT) and/or uridinenucleotides. In other embodiments, one or more of the nucleotides in the3′ overhangs on one or both sides of the double-stranded region comprisemodified nucleotides. Non-limiting examples of modified nucleotides aredescribed above and include 2′OMe nucleotides, 2′-deoxy-2′F nucleotides,2′-deoxy nucleotides, 2′-O-2-MOE nucleotides, LNA nucleotides, andmixtures thereof. In some embodiments, one, two, three, four, or morenucleotides in the 3′ overhangs present on the sense and/or antisensestrand of the siRNA comprise 2′OMe nucleotides (e.g., 2′OMe purineand/or pyrimidine nucleotides) such as, for example, 2′OMe-guanosinenucleotides, 2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides,2′OMe-cytosine nucleotides, and mixtures thereof.

The siRNA may comprise at least one or a cocktail (e.g., at least two,three, four, five, six, seven, eight, nine, ten, or more) of unmodifiedand/or modified siRNA sequences that silence target gene expression. Thecocktail of siRNA may comprise sequences which are directed to the sameregion or domain (e.g., a “hot spot”) and/or to different regions ordomains of one or more target genes. In certain instances, one or more(e.g., at least two, three, four, five, six, seven, eight, nine, ten, ormore) modified siRNA that silence target gene expression are present ina cocktail. In certain other instances, one or more (e.g., at least two,three, four, five, six, seven, eight, nine, ten, or more) unmodifiedsiRNA sequences that silence target gene expression are present in acocktail.

In some embodiments, the antisense strand of the siRNA moleculecomprises or consists of a sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence ora portion thereof. In other embodiments, the antisense strand of thesiRNA molecule comprises or consists of a sequence that is 100%complementary to the target sequence or a portion thereof. In furtherembodiments, the antisense strand of the siRNA molecule comprises orconsists of a sequence that specifically hybridizes to the targetsequence or a portion thereof.

In further embodiments, the sense strand of the siRNA molecule comprisesor consists of a sequence that is at least about 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the target sequence or a portionthereof. In additional embodiments, the sense strand of the siRNAmolecule comprises or consists of a sequence that is 100% identical tothe target sequence or a portion thereof.

In the lipid particles of the invention (e.g., LNP comprising aninterfering RNA, such as siRNA, or LNP comprising one or more mRNAmolecules), the cationic lipid may comprise, e.g., one or more of thefollowing: 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2”), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane(DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane(DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane(DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane(DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane(DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane(DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), ormixtures thereof. In certain embodiments, the cationic lipid is DLinDMA,DLin-K-C2-DMA (“XTC2”), or mixtures thereof.

The synthesis of cationic lipids such as DLin-K-C2-DMA (“XTC2”),DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well asadditional cationic lipids, is described in U.S. Provisional ApplicationNo. 61/104,212, filed Oct. 9, 2008, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Thesynthesis of cationic lipids such as DLin-K-DMA, DLin-C-DAP, DLin-DAC,DLin-MA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl,DLin-MPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationiclipids, is described in PCT Application No. PCT/US08/88676, filed Dec.31, 2008, the disclosure of which is herein incorporated by reference inits entirety for all purposes. The synthesis of cationic lipids such asCLinDMA, as well as additional cationic lipids, is described in U.S.Patent Publication No. 20060240554, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

In some embodiments, the cationic lipid may comprise from about 30 mol %to about 90 mol %, from about 30 mol % to about 85 mol %, from about 30mol % to about 80 mol %, from about 30 mol % to about 75 mol %, fromabout 30 mol % to about 70 mol %, from about 30 mol % to about 65 mol %,or from about 30 mol % to about 60 mol % of the total lipid present inthe particle.

In some embodiments, the cationic lipid may comprise from about 40 mol %to about 90 mol %, from about 40 mol % to about 85 mol %, from about 40mol % to about 80 mol %, from about 40 mol % to about 75 mol %, fromabout 40 mol % to about 70 mol %, from about 40 mol % to about 65 mol %,or from about 40 mol % to about 60 mol % of the total lipid present inthe particle.

In other embodiments, the cationic lipid may comprise from about 55 mol% to about 90 mol %, from about 55 mol % to about 85 mol %, from about55 mol % to about 80 mol %, from about 55 mol % to about 75 mol %, fromabout 55 mol % to about 70 mol %, or from about 55 mol % to about 65 mol% of the total lipid present in the particle.

In yet other embodiments, the cationic lipid may comprise from about 60mol % to about 90 mol %, from about 60 mol % to about 85 mol %, fromabout 60 mol % to about 80 mol %, from about 60 mol % to about 75 mol %,or from about 60 mol % to about 70 mol % of the total lipid present inthe particle.

In still yet other embodiments, the cationic lipid may comprise fromabout 65 mol % to about 90 mol %, from about 65 mol % to about 85 mol %,from about 65 mol % to about 80 mol %, or from about 65 mol % to about75 mol % of the total lipid present in the particle.

In further embodiments, the cationic lipid may comprise from about 70mol % to about 90 mol %, from about 70 mol % to about 85 mol %, fromabout 70 mol % to about 80 mol %, from about 75 mol % to about 90 mol %,from about 75 mol % to about 85 mol %, or from about 80 mol % to about90 mol % of the total lipid present in the particle.

In additional embodiments, the cationic lipid may comprise (at least)about 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, or 90 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle.

In the lipid particles of the invention (e.g., LNP comprising aninterfering RNA, such as siRNA; or LNP comprising one or more mRNAmolecules), the non-cationic lipid may comprise, e.g., one or moreanionic lipids and/or neutral lipids. In some embodiments, thenon-cationic lipid comprises one of the following neutral lipidcomponents: (1) cholesterol or a derivative thereof (2) a phospholipid;or (3) a mixture of a phospholipid and cholesterol or a derivativethereof.

Examples of cholesterol derivatives include, but are not limited to,cholestanol, cholestanone, cholestenone, coprostanol,cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether,and mixtures thereof. The synthesis of cholesteryl-2′-hydroxyethyl etheris described herein.

The phospholipid may be a neutral lipid including, but not limited to,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine(EPC), and mixtures thereof. In certain embodiments, the phospholipid isDPPC, DSPC, or mixtures thereof.

In some embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol% to about 60 mol %, from about 25 mol % to about 60 mol %, from about30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, fromabout 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %,from about 25 mol % to about 55 mol %, from about 30 mol % to about 55mol %, from about 13 mol % to about 50 mol %, from about 15 mol % toabout 50 mol % or from about 20 mol % to about 50 mol % of the totallipid present in the particle. When the non-cationic lipid is a mixtureof a phospholipid and cholesterol or a cholesterol derivative, themixture may comprise up to about 40, 50, or 60 mol % of the total lipidpresent in the particle.

In other embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 49.5 mol %, from about 13 mol % to about 49.5 mol %, from about 15mol % to about 49.5 mol %, from about 20 mol % to about 49.5 mol %, fromabout 25 mol % to about 49.5 mol %, from about 30 mol % to about 49.5mol %, from about 35 mol % to about 49.5 mol %, or from about 40 mol %to about 49.5 mol % of the total lipid present in the particle.

In yet other embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 45 mol %, from about 13 mol % to about 45 mol %, from about 15 mol% to about 45 mol %, from about 20 mol % to about 45 mol %, from about25 mol % to about 45 mol %, from about 30 mol % to about 45 mol %, orfrom about 35 mol % to about 45 mol % of the total lipid present in theparticle.

In still yet other embodiments, the non-cationic lipid (e.g., one ormore phospholipids and/or cholesterol) may comprise from about 10 mol %to about 40 mol %, from about 13 mol % to about 40 mol %, from about 15mol % to about 40 mol %, from about 20 mol % to about 40 mol %, fromabout 25 mol % to about 40 mol %, or from about 30 mol % to about 40 mol% of the total lipid present in the particle.

In further embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 35 mol %, from about 13 mol % to about 35 mol %, from about 15 mol% to about 35 mol %, from about 20 mol % to about 35 mol %, or fromabout 25 mol % to about 35 mol % of the total lipid present in theparticle.

In yet further embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 30 mol %, from about 13 mol % to about 30 mol %, from about 15 mol% to about 30 mol %, from about 20 mol % to about 30 mol %, from about10 mol % to about 25 mol %, from about 13 mol % to about 25 mol %, orfrom about 15 mol % to about 25 mol % of the total lipid present in theparticle.

In additional embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise (at least) about 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle.

In certain embodiments, the non-cationic lipid comprises cholesterol ora derivative thereof of from about 31.5 mol % to about 42.5 mol % of thetotal lipid present in the particle. As a non-limiting example, aphospholipid-free lipid particle of the invention may comprisecholesterol or a derivative thereof at about 37 mol % of the total lipidpresent in the particle. In other embodiments, a phospholipid-free lipidparticle of the invention may comprise cholesterol or a derivativethereof of from about 30 mol % to about 45 mol %, from about 30 mol % toabout 40 mol %, from about 30 mol % to about 35 mol %, from about 35 mol% to about 45 mol %, from about 40 mol % to about 45 mol %, from about32 mol % to about 45 mol %, from about 32 mol % to about 42 mol %, fromabout 32 mol % to about 40 mol %, from about 34 mol % to about 45 mol %,from about 34 mol % to about 42 mol %, from about 34 mol % to about 40mol %, or about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, or 45 mol % (or any fraction thereof or range therein) of the totallipid present in the particle.

In certain other embodiments, the non-cationic lipid comprises a mixtureof: (i) a phospholipid of from about 4 mol % to about 10 mol % of thetotal lipid present in the particle; and (ii) cholesterol or aderivative thereof of from about 30 mol % to about 40 mol % of the totallipid present in the particle. As a non-limiting example, a lipidparticle comprising a mixture of a phospholipid and cholesterol maycomprise DPPC at about 7 mol % and cholesterol at about 34 mol % of thetotal lipid present in the particle. In other embodiments, thenon-cationic lipid comprises a mixture of: (i) a phospholipid of fromabout 3 mol % to about 15 mol %, from about 4 mol % to about 15 mol %,from about 4 mol % to about 12 mol %, from about 4 mol % to about 10 mol%, from about 4 mol % to about 8 mol %, from about 5 mol % to about 12mol %, from about 5 mol % to about 9 mol %, from about 6 mol % to about12 mol %, from about 6 mol % to about 10 mol %, or about 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle; and (ii)cholesterol or a derivative thereof of from about 25 mol % to about 45mol %, from about 30 mol % to about 45 mol %, from about 25 mol % toabout 40 mol %, from about 30 mol % to about 40 mol %, from about 25 mol% to about 35 mol %, from about 30 mol % to about 35 mol %, from about35 mol % to about 45 mol %, from about 40 mol % to about 45 mol %, fromabout 28 mol % to about 40 mol %, from about 28 mol % to about 38 mol %,from about 30 mol % to about 38 mol %, from about 32 mol % to about 36mol %, or about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, or 45 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In further embodiments, the non-cationic lipid comprises a mixture of:(i) a phospholipid of from about 10 mol % to about 30 mol % of the totallipid present in the particle; and (ii) cholesterol or a derivativethereof of from about 10 mol % to about 30 mol % of the total lipidpresent in the particle. As a non-limiting example, a lipid particlecomprising a mixture of a phospholipid and cholesterol may comprise DPPCat about 20 mol % and cholesterol at about 20 mol % of the total lipidpresent in the particle. In other embodiments, the non-cationic lipidcomprises a mixture of: (i) a phospholipid of from about 10 mol % toabout 30 mol %, from about 10 mol % to about 25 mol %, from about 10 mol% to about 20 mol %, from about 15 mol % to about 30 mol %, from about20 mol % to about 30 mol %, from about 15 mol % to about 25 mol %, fromabout 12 mol % to about 28 mol %, from about 14 mol % to about 26 mol %,or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 mol % (or any fraction thereof or range therein)of the total lipid present in the particle; and (ii) cholesterol or aderivative thereof of from about 10 mol % to about 30 mol %, from about10 mol % to about 25 mol %, from about 10 mol % to about 20 mol %, fromabout 15 mol % to about 30 mol %, from about 20 mol % to about 30 mol %,from about 15 mol % to about 25 mol %, from about 12 mol % to about 28mol %, from about 14 mol % to about 26 mol %, or about 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30mol % (or any fraction thereof or range therein) of the total lipidpresent in the particle.

In certain instances, the PEG-conjugated lipid of formula (I) maycomprise from about 0.1 mol % to about 2 mol %, from about 0.5 mol % toabout 2 mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol %to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about0.8 mol % to about 1.7 mol %, from about 1 mol % to about 1.8 mol %,from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about1.7 mol %, from about 1.3 mol % to about 1.6 mol %, from about 1.4 mol %to about 1.5 mol %, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or 2 mol % (or any fraction thereof or range therein) of the totallipid present in the particle.

In the lipid particles of the invention, the active agent or therapeuticagent may be fully encapsulated within the lipid portion of theparticle, thereby protecting the active agent or therapeutic agent fromenzymatic degradation. In some embodiments, a LNP comprising a nucleicacid, such as an interfering RNA (e.g., siRNA) or mRNA, is fullyencapsulated within the lipid portion of the particle, therebyprotecting the nucleic acid from nuclease degradation. In certaininstances, the nucleic acid in the LNP is not substantially degradedafter exposure of the particle to a nuclease at 37° C. for at leastabout 20, 30, 45, or 60 minutes. In certain other instances, the nucleicacid in the LNP is not substantially degraded after incubation of theparticle in serum at 37° C. for at least about 30, 45, or 60 minutes orat least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, or 36 hours. In other embodiments, the active agentor therapeutic agent (e.g., nucleic acid, such as siRNA or mRNA) iscomplexed with the lipid portion of the particle. One of the benefits ofthe formulations of the present invention is that the lipid particlecompositions are substantially non-toxic to mammals such as humans.

The term “fully encapsulated” indicates that the active agent ortherapeutic agent in the lipid particle is not significantly degradedafter exposure to serum or a nuclease or protease assay that wouldsignificantly degrade free DNA, RNA, or protein. In a fully encapsulatedsystem, typically less than about 25% of the active agent or therapeuticagent in the particle is degraded in a treatment that would normallydegrade 100% of free active agent or therapeutic agent, for example,less than about 10%, or less than about 5% of the active agent ortherapeutic agent in the particle is degraded. In the context of nucleicacid therapeutic agents, full encapsulation may be determined by anOligreen® assay. Oligreen® is an ultra-sensitive fluorescent nucleicacid stain for quantitating oligonucleotides and single-stranded DNA orRNA in solution (available from Invitrogen Corporation; Carlsbad,Calif.). “Fully encapsulated” also indicates that the lipid particlesare serum-stable, that is, that they do not rapidly decompose into theircomponent parts upon in vivo administration.

In another aspect, the present invention provides a lipid particle(e.g., LNP) composition comprising a plurality of lipid particles. Insome embodiments, the active agent or therapeutic agent (e.g., nucleicacid) is fully encapsulated within the lipid portion of the lipidparticles (e.g., LNP), such that from about 30% to about 100%, fromabout 40% to about 100%, from about 50% to about 100%, from about 60% toabout 100%, from about 70% to about 100%, from about 80% to about 100%,from about 90% to about 100%, from about 30% to about 95%, from about40% to about 95%, from about 50% to about 95%, from about 60% to about95%, %, from about 70% to about 95%, from about 80% to about 95%, fromabout 85% to about 95%, from about 90% to about 95%, from about 30% toabout 90%, from about 40% to about 90%, from about 50% to about 90%,from about 60% to about 90%, from about 70% to about 90%, from about 80%to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%(or any fraction thereof or range therein) of the lipid particles (e.g.,LNP) have the active agent or therapeutic agent encapsulated therein.

Typically, the lipid particles (e.g., LNP) of the invention have alipid:active agent (e.g., lipid:nucleic acid) ratio (mass/mass ratio) offrom about 1 to about 100. In some instances, the lipid:active agent(e.g., lipid:nucleic acid) ratio (mass/mass ratio) ranges from about 1to about 50, from about 2 to about 25, from about 3 to about 20, fromabout 4 to about 15, or from about 5 to about 10. In some embodiments,the lipid particles of the invention have a lipid:active agent (e.g.,lipid:nucleic acid) ratio (mass/mass ratio) of from about 5 to about 15,e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any fractionthereof or range therein).

Typically, the lipid particles (e.g., LNP) of the invention have a meandiameter of from about 40 nm to about 150 nm. In some embodiments, thelipid particles (e.g., LNP) of the invention have a mean diameter offrom about 40 nm to about 130 nm, from about 40 nm to about 120 nm, fromabout 40 nm to about 100 nm, from about 50 nm to about 120 nm, fromabout 50 nm to about 100 nm, from about 60 nm to about 120 nm, fromabout 60 nm to about 110 nm, from about 60 nm to about 100 nm, fromabout 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about70 nm to about 120 nm, from about 70 nm to about 110 nm, from about 70nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm toabout 80 nm, or less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm(or any fraction thereof or range therein).

In one specific embodiment of the invention, the LNP comprises: (a) oneor more unmodified and/or modified nucleic acid molecules (e.g.,interfering RNA that silence target gene expression, such as siRNA,aiRNA, miRNA; or mRNA that result in target protein expression); (b) acationic lipid comprising from about 56.5 mol % to about 66.5 mol % ofthe total lipid present in the particle; (c) a non-cationic lipidcomprising from about 31.5 mol % to about 42.5 mol % of the total lipidpresent in the particle; and (d) a conjugated lipid that inhibitsaggregation of particles comprising from about 1 mol % to about 2 mol %of the total lipid present in the particle. This specific embodiment ofLNP is generally referred to herein as the “1:62” formulation. In oneembodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA (“XTC2”), thenon-cationic lipid is cholesterol, and the conjugated lipid is a PEG-DAAconjugate. Although these are typical embodiments of the 1:62formulation, those of skill in the art will appreciate that othercationic lipids, non-cationic lipids (including other cholesterolderivatives), and conjugated lipids can be used in the 1:62 formulationas described herein.

In another specific embodiment of the invention, the LNP comprises: (a)one or more unmodified and/or modified nucleic acid molecules (e.g.,interfering RNA that silence target gene expression, such as siRNA,aiRNA, miRNA; or mRNA that result in target protein expression); (b) acationic lipid comprising from about 52 mol % to about 62 mol % of thetotal lipid present in the particle; (c) a non-cationic lipid comprisingfrom about 36 mol % to about 47 mol % of the total lipid present in theparticle; and (d) a PEG-conjugated lipid of formula (I) comprising fromabout 1 mol % to about 2 mol % of the total lipid present in theparticle.

In one embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA(“XTC2”), the non-cationic lipid is a mixture of a phospholipid (such asDPPC) and cholesterol, wherein the phospholipid comprises from about 5mol % to about 9 mol % of the total lipid present in the particle (e.g.,about 7.1 mol %) and the cholesterol (or cholesterol derivative)comprises from about 32 mol % to about 37 mol % of the total lipidpresent in the particle (e.g., about 34.3 mol %), and the PEG-lipid is acompound of formula (I).

In another embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA(“XTC2”), the non-cationic lipid is a mixture of a phospholipid (such asDPPC) and cholesterol, wherein the phospholipid comprises from about 15mol % to about 25 mol % of the total lipid present in the particle(e.g., about 20 mol %) and the cholesterol (or cholesterol derivative)comprises from about 15 mol % to about 25 mol % of the total lipidpresent in the particle (e.g., about 20 mol %), and the PEG-lipid is acompound of formula (I).

In one embodiment, the 1:62 LNP formulation is a three-component systemwhich is phospholipid-free and comprises about 1.5 mol % PEG-conjugatedlipids of formula (I), about 61.5 mol % DLinDMA (or XTC2), and about36.9 mol % cholesterol (or derivative thereof). In other embodiments,the 1:57 LNP formulation is a four-component system which comprisesabout 1.4 mol PEG-conjugated lipids of formula (I), about 57.1 mol %DLinDMA (or XTC2), about 7.1 mol % DPPC, and about 34.3 mol %cholesterol (or derivative thereof). In yet another embodiment, the LNPformulation is a four-component system which comprises about 1.4 molPEG-conjugated lipids of formula (I), about 57.1 mol % DLinDMA (orXTC2), about 20 mol % DPPC, and about 20 mol % cholesterol (orderivative thereof). It should be understood that these LNP formulationsare target formulations, and that the amount of lipid (both cationic andnon-cationic) present and the amount of lipid conjugate present in theLNP formulations may vary.

In a further aspect, the present invention provides a method forintroducing one or more active agents or therapeutic agents (e.g.,nucleic acid) into a cell, comprising contacting the cell with a lipidparticle (e.g., LNP) described herein. In one embodiment, the cell is ina mammal and the mammal is a human. In another embodiment, the presentinvention provides a method for the in vivo delivery of one or moreactive agents or therapeutic agents (e.g., nucleic acid), comprisingadministering to a mammalian subject a lipid particle (e.g., LNP)described herein. In one embodiment, the mode of administrationincludes, but is not limited to, oral, intranasal, intravenous,intraperitoneal, intramuscular, intra-articular, intralesional,intratracheal, subcutaneous, and intradermal. Typically, the mammaliansubject is a human.

In one embodiment, at least about 5%, 10%, 15%, 20%, or 25% of the totalinjected dose of the lipid particles (e.g., LNP) is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In other embodiments,more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% ofthe total injected dose of the lipid particles (e.g., LNP) is present inplasma about 8, 12, 24, 36, or 48 hours after injection. In certaininstances, more than about 10% of a plurality of the particles ispresent in the plasma of a mammal about 1 hour after administration. Incertain other instances, the presence of the lipid particles (e.g., LNP)is detectable at least about 1 hour after administration of theparticle. In certain embodiments, the presence of an active agent ortherapeutic agent, such as an interfering RNA (e.g., siRNA) or mRNA isdetectable in cells of the at about 8, 12, 24, 36, 48, 60, 72 or 96hours after administration (e.g., lung, liver, tumor, or at a site ofinflammation). In other embodiments, downregulation of expression of atarget sequence by an active agent or therapeutic agent, such as aninterfering RNA (e.g., siRNA) is detectable at about 8, 12, 24, 36, 48,60, 72 or 96 hours after administration. In yet other embodiments,downregulation of expression of a target sequence by an active agent ortherapeutic agent such as an interfering RNA (e.g., siRNA) occurs intumor cells or in cells at a site of inflammation. In furtherembodiments, the presence or effect of an active agent or therapeuticagent such as an interfering RNA (e.g., siRNA) in cells at a siteproximal or distal to the site of administration or in cells of thelung, liver, or a tumor is detectable at about 12, 24, 48, 72, or 96hours, or at about 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28days after administration. In other embodiments, upregulation ofexpression of a target sequence by an active agent or therapeutic agent,such as an mRNA or self-amplifying RNA is detectable at about 8, 12, 24,36, 48, 60, 72 or 96 hours after administration. In yet otherembodiments, upregulation of expression of a target sequence by anactive agent or therapeutic agent such as an mRNA or self-amplifying RNAoccurs in tumor cells or in cells at a site of inflammation. In furtherembodiments, the presence or effect of an active agent or therapeuticagent such as an mRNA or self-amplifying RNA in cells at a site proximalor distal to the site of administration or in cells of the lung, liver,or a tumor is detectable at about 12, 24, 48, 72, or 96 hours, or atabout 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days afteradministration. In additional embodiments, the lipid particles (e.g.,LNP) of the invention are administered parenterally orintraperitoneally. In embodiments, the lipid particles (e.g., LNP) ofthe invention are administered intramuscularly.

In some embodiments, the lipid particles (e.g., LNP) of the inventionare useful in methods for the therapeutic delivery of one or morenucleic acids comprising an interfering RNA sequence (e.g., siRNA). Inparticular, one object of this invention to provide in vitro and in vivomethods for treatment of a disease or disorder in a mammal (e.g., arodent such as a mouse or a primate such as a human, chimpanzee, ormonkey) by downregulating or silencing the transcription and/ortranslation of one or more target nucleic acid sequences or genes ofinterest. As a non-limiting example, the methods of the invention areuseful for in vivo delivery of interfering RNA (e.g., siRNA) to theliver and/or tumor of a mammalian subject. In certain embodiments, thedisease or disorder is associated with expression and/or overexpressionof a gene and expression or overexpression of the gene is reduced by theinterfering RNA (e.g., siRNA). In certain other embodiments, atherapeutically effective amount of the lipid particle (e.g., LNP) maybe administered to the mammal. In some instances, an interfering RNA(e.g., siRNA) is formulated into a LNP, and the particles areadministered to patients requiring such treatment. In other instances,cells are removed from a patient, the interfering RNA (e.g., siRNA) isdelivered in vitro (e.g., using a LNP described herein), and the cellsare reinjected into the patient.

In an additional aspect, the present invention provides lipid particles(e.g., LNP) comprising asymmetrical interfering RNA (aiRNA) moleculesthat silence the expression of a target gene and methods of using suchparticles to silence target gene expression.

In one embodiment, the aiRNA molecule comprises a double-stranded(duplex) region of about 10 to about 25 (base paired) nucleotides inlength, wherein the aiRNA molecule comprises an antisense strandcomprising 5′ and 3′ overhangs, and wherein the aiRNA molecule iscapable of silencing target gene expression.

In certain instances, the aiRNA molecule comprises a double-stranded(duplex) region of about 12-20, 12-19, 12-18, 13-17, or 14-17 (basepaired) nucleotides in length, more typically 12, 13, 14, 15, 16, 17,18, 19, or 20 (base paired) nucleotides in length. In certain otherinstances, the 5′ and 3′ overhangs on the antisense strand comprisesequences that are complementary to the target RNA sequence, and mayoptionally further comprise nontargeting sequences. In some embodiments,each of the 5′ and 3′ overhangs on the antisense strand comprises orconsists of one, two, three, four, five, six, seven, or morenucleotides.

In other embodiments, the aiRNA molecule comprises modified nucleotidesselected from the group consisting of 2′OMe nucleotides, 2′Fnucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNAnucleotides, and mixtures thereof. In one embodiment, the aiRNA moleculecomprises 2′OMe nucleotides. As a non-limiting example, the 2′OMenucleotides may be selected from the group consisting of 2′OMe-guanosinenucleotides, 2′OMe-uridine nucleotides, and mixtures thereof.

In a related aspect, the present invention provides lipid particles(e.g., LNP) comprising microRNA (miRNA) molecules that silence theexpression of a target gene and methods of using such compositions tosilence target gene expression.

In one embodiment, the miRNA molecule comprises about 15 to about 60nucleotides in length, wherein the miRNA molecule is capable ofsilencing target gene expression.

In certain instances, the miRNA molecule comprises about 15-50, 15-40,or 15-30 nucleotides in length, more typically about 15-25 or 19-25nucleotides in length, and are typically about 20-24, 21-22, or 21-23nucleotides in length. In one embodiment, the miRNA molecule is a maturemiRNA molecule targeting an RNA sequence of interest.

In some embodiments, the miRNA molecule comprises modified nucleotidesselected from the group consisting of 2′OMe nucleotides, 2′Fnucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNAnucleotides, and mixtures thereof. In one embodiment, the miRNA moleculecomprises 2′OMe nucleotides. As a non-limiting example, the 2′OMenucleotides may be selected from the group consisting of 2′OMe-guanosinenucleotides, 2′OMe-uridine nucleotides, and mixtures thereof.

In some embodiments, the lipid particles (e.g., LNP) of the inventionare useful in methods for the therapeutic delivery of one or more mRNAmolecules. In particular, it is one object of this invention to providein vitro and in vivo methods for treatment of a disease or disorder in amammal (e.g., a rodent such as a mouse or a primate such as a human,chimpanzee, or monkey) through the expression of one or more targetproteins. As a non-limiting example, the methods of the invention areuseful for in vivo delivery of one or more mRNA molecules to a mammaliansubject. In certain other embodiments, a therapeutically effectiveamount of the lipid particle (e.g., LNP) may be administered to themammal. In some instances, one or more mRNA molecules are formulatedinto a LNP, and the particles are administered to patients requiringsuch treatment. In other instances, cells are removed from a patient,one or more mRNA molecules are delivered in vitro (e.g., using a LNPdescribed herein), and the cells are reinjected into the patient.

In other embodiments, the mRNA molecule comprises modified nucleotidesselected from the group consisting of 2′OMe nucleotides, 2′Fnucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNAnucleotides, and mixtures thereof. In a related aspect, the presentinvention provides lipid particles (e.g., LNP) comprising microRNA(miRNA) molecules that silence the expression of a target gene andmethods of using such compositions to silence target gene expression.

As such, the lipid particles of the invention (e.g., LNP) areadvantageous and suitable for use in the administration of active agentsor therapeutic agents, such as nucleic acid (e.g., interfering RNA suchas siRNA, aiRNA, and/or miRNA; or mRNA) to a subject (e.g., a mammalsuch as a human) because they are stable in circulation, of a sizerequired for pharmacodynamic behavior resulting in access toextravascular sites, and are capable of reaching target cellpopulations.

In one embodiment A is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,or (C₂-C₆)alkynyl, wherein any (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,(C₂-C₆)alkenyl, and (C₂-C₆)alkynyl is substituted with one or moreanionic precursor groups, and wherein any (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl, and (C₂-C₆)alkynyl is optionallysubstituted with one or more groups independently selected from thegroup consisting of halo, hydroxyl, (C₁-C₃)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₃)alkoxycarbonyl, (C₁-C₃)alkylthio, or (C₂-C₃)alkanoyloxy.

In one embodiment A is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,or (C₂-C₆)alkynyl, wherein any (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,(C₂-C₆)alkenyl, and (C₂-C₆)alkynyl is substituted with one or moreanionic precursor groups.

In one embodiment A is (C₁-C₆)alkyl that is substituted with one or moreanionic precursor groups, and is optionally substituted with one or moregroups independently selected from the group consisting of halo,hydroxyl, (C₁-C₃)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₁-C₃)alkylthio, or (C₂-C₃)alkanoyloxy.

In one embodiment A is (C₁-C₆)alkyl that is substituted with one or moreanionic precursor groups.

In one embodiment A is substituted with one anionic precursor group.

In one embodiment A is substituted with two anionic precursor groups.

In one embodiment A is substituted with three anionic precursor groups.

In one embodiment each anionic precursor group is selected from thegroup consisting of —CO₂H, —O—P(═O)(OH)₂, —OS(═O)₂(OH), —O—S(═O)(OH),and —B(OH)₂.

In one embodiment each anionic precursor group is —CO₂H.

In one embodiment B is a polyethylene glycol chain having an averagemolecular weight ranging of about 500 daltons to about 5,000 daltons.

In one embodiment B is a polyethylene glycol chain having an averagemolecular weight ranging of about 500 daltons to about 1,000 daltons.

In one embodiment B is a polyethylene glycol chain having an averagemolecular weight ranging of about 750 daltons to about 3,000 daltons.

In one embodiment B is a polyethylene glycol chain having an averagemolecular weight ranging of about 1000 daltons to about 2000 daltons.

In one embodiment B is a polyethylene glycol chain having an averagemolecular weight range of about 2,000 daltons.

In one embodiment B is linked to C directly.

In one embodiment B is linked to C directly via a linker moiety.

In one embodiment B is linked to C through a group selected from thegroup consisting of —C(O)NH—, —NR—, —C(O)—, —NHC(O)O—, —NHC(O)NH—,—S—S—, —O—, —(O)CCH₂CH₂C(O)—, and —NHC(O)CH₂CH₂C(O)NH—, wherein R is Hor (C₁-C₆)alkyl.

In one embodiment A is linked to B directly.

In one embodiment A is linked to B directly via a linker moiety.

In one embodiment A is linked to B through a group selected from thegroup consisting of —C(O)NH—, —NR—, —C(O)—, —NHC(O)O—, —NHC(O)NH—,—S—S—, —O—, —(O)CCH₂CH₂C(O)—, and —NHC(O)CH₂CH₂C(O)NH—.

In one embodiment L is —C(O)NR^(b)—, —NR^(b)—, —C(O)—, —NR^(b)C(O)O—,—NR^(b)C(O)NR^(b)—, —S—S—, —O—, —(O)CCH₂CH₂C(O)—, or—NHC(O)CH₂CH₂C(O)NH—.

In one embodiment L is —NR^(b)—.

In one embodiment R^(a) is a branched (C₁₀-C₅₀)alkyl or branched(C₁₀-C₅₀)alkenyl, wherein two or more carbon atoms of the branched(C₁₀-C₅₀)alkyl or branched (C₁₀-C₅₀)alkenyl have been replaced with —O—.

In one embodiment R^(a) is a branched (C₁₀-C₅₀)alkyl or branched(C₁₀-C₅₀)alkenyl, wherein two of the branched (C₁₀-C₅₀)alkyl or branched(C₁₀-C₅₀)alkenyl have been replaced with —O—.

In one embodiment R^(a) is a branched (C₁₀-C₅₀)alkyl, wherein two of thebranched (C₁₀-C₅₀)alkyl or branched (C₁₀-C₅₀)alkenyl have been replacedwith —O—.

In one embodiment each R^(b) is independently H.

In one embodiment C has the following structure:

wherein:

R¹ and R² are each independently (C₁₀-C₂₀)alkyl or (C₁₀-C₂₀)alkenyl;

M is a direct bond or a divalent (C₁-C₅)alkyl;

L is selected from the group consisting of a direct bond, —C(O)O—,—C(O)NR^(b)—, —NR^(b)—, —C(O)—, —NR^(b)C(O)O—, —NR^(b)C(O)NR^(b)—,—S—S—, —O—, —(O)CCH₂CH₂C(O)—, and —NHC(O)CH₂CH₂C(O)NH—; and

each R^(b) is independently H or (C₁-C₆)alkyl.

In one embodiment L is selected from the group consisting of—C(O)NR^(b)—, —NR^(b)—, —C(O)—, —NR^(b)C(O)O—, —NR^(b)C(O)NR^(b)—,—S—S—, —O—, —(O)CCH₂CH₂C(O)—, and —NHC(O)CH₂CH₂C(O)NH—.

In one embodiment L is —NR^(b)—.

In one embodiment M is a direct bond.

In one embodiment M is a divalent (C₁-C₅)alkyl.

In one embodiment R¹ is selected from the group consisting of lauryl(C12), myristyl (C14), palmityl (C16), stearyl (C18) and icosyl (C20).

In one embodiment R² is selected from the group consisting of lauryl(C12), myristyl (C14), palmityl (C16), stearyl (C18) and icosyl (C20).

In one embodiment R¹ and R² are the same.

In one embodiment R¹ and R² are both lauric (C12).

In one embodiment R¹ and R² are both myristyl (C14).

In one embodiment R¹ and R² are both palmityl (C16).

In one embodiment R¹ and R² are both stearyl (C18).

In one embodiment the non-cationic lipid is a member selected from thegroup consisting of dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC),palmitoyloleyolphosphatidylglycerol (POPG), cholesterol, and a mixturethereof.

In one embodiment the non-cationic lipid is an anionic lipid.

In one embodiment the non-cationic lipid is a neutral lipid.

In one embodiment the cationic lipid comprises from about 5% to about90% of the total lipid present in said particle.

In one embodiment the cationic lipid comprises from about 15% to about90% of the total lipid present in said particle.

In one embodiment the cationic lipid comprises from about 25% to about90% of the total lipid present in said particle.

In one embodiment the cationic lipid comprises from about 5% to about80% of the total lipid present in said particle.

In one embodiment the cationic lipid comprises from about 15% to about70% of the total lipid present in said particle.

In one embodiment the cationic lipid comprises from about 25% to about60% of the total lipid present in said particle.

In one embodiment the cationic lipid comprises from about 40% to about60% of the total lipid present in said particle.

In one embodiment the non-cationic lipid is DSPC.

In one embodiment the non-cationic lipid comprises cholesterol.

In one embodiment the cholesterol comprises from about 10% to about 60%of the total lipid present in said particle.

In one embodiment the cholesterol comprises from about 20% to about 45%of the total lipid present in said particle.

In one embodiment the nucleic acid is DNA.

In one embodiment the nucleic acid is RNA.

In one embodiment the nucleic acid is selected from the group consistingof small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpinRNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA),mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, guide RNAfor gene editing systems, DNA, plasmids, antisense oligonucleotides, andcombinations thereof.

In one embodiment the nucleic acid is a ribozyme.

In one embodiment the nucleic acid is a small interfering RNA (siRNA).

In one embodiment the nucleic acid is mRNA, and wherein the mRNA encodesa therapeutic product of interest. In one embodiment the therapeuticproduct of interest is a peptide or protein. In one embodiment thetherapeutic product of interest is a vaccine antigen.

In one embodiment the nucleic acid-lipid particle is not substantiallydegraded after exposure of said particle to a nuclease at 37° C. for 20minutes.

In one embodiment the nucleic acid-lipid particle is not substantiallydegraded after incubation of said particle in serum at 37° C. for 30minutes.

In one embodiment the one or more nucleic acid molecules are fullyencapsulated in said nucleic acid-lipid particle.

In one embodiment the nucleic acid-lipid particle has a lipid:nucleicacid mass ratio of from about 5 to about 30.

In one embodiment the nucleic acid-lipid particle has a lipid:nucleicacid mass ratio of from about 5 to about 15.

In one embodiment the nucleic acid is mRNA and wherein the nucleicacid-lipid particle has a lipid:nucleic acid mass ratio of from about 15to about 25.

In one embodiment the invention provides a pharmaceutical compositioncomprising: about 1.5% of total lipid of the compound:

about 50.0% of total lipid of the compound:

about 38.5% of total lipid of cholesterol; and

about 10.0% of total lipid of DSPC.

In one embodiment the invention provides a composition comprising:

1.5% of total lipid of the compound:

50.0% of total lipid of the compound:

38.5% of total lipid of cholesterol; and

10.0% of total lipid of DSPC.

In one embodiment the invention provides a composition comprising

about 2.0% of total lipid of the compound:

about 40.0% of total lipid of the compound:

about 48.5% of total lipid of cholesterol; and

about 10.0% of total lipid of DSPC.

In one embodiment the invention provides a composition comprising

2.0% of total lipid of the compound:

40.0% of total lipid of the compound:

48.5% of total lipid of cholesterol; and

10.0% of total lipid of DSPC.

In one embodiment the invention provides a composition comprising

about 1.6% of total lipid of the compound:

about 54.9% of total lipid of the compound:

about 32.8% of total lipid of cholesterol; and

about 10.0% of total lipid of DSPC.

In one embodiment the invention provides a composition comprising

1.6% of total lipid of the compound:

54.9% of total lipid of the compound:

32.8% of total lipid of cholesterol; and

10.0% of total lipid of DSPC.

II. ACTIVE AGENTS

Active agents (e.g., therapeutic agents) include any molecule orcompound capable of exerting a desired effect on a cell, tissue, organ,or subject. Such effects may be, e.g., biological, physiological, and/orcosmetic. Active agents may be any type of molecule or compoundincluding, but not limited to, nucleic acids, peptides, polypeptides,small molecules, and mixtures thereof. Non-limiting examples of nucleicacids include interfering RNA molecules (e.g., siRNA, aiRNA, miRNA),antisense oligonucleotides, mRNA, self-amplifying RNA, plasmids,ribozymes, immunostimulatory oligonucleotides, and mixtures thereof.Examples of peptides or polypeptides include, without limitation,antibodies (e.g., polyclonal antibodies, monoclonal antibodies, antibodyfragments; humanized antibodies, recombinant antibodies, recombinanthuman antibodies, Primatized™ antibodies), cytokines, growth factors,apoptotic factors, differentiation-inducing factors, cell-surfacereceptors and their ligands, hormones, and mixtures thereof. Examples ofsmall molecules include, but are not limited to, small organic moleculesor compounds such as any conventional agent or drug known to those ofskill in the art.

In some embodiments, the active agent is a therapeutic agent, or a saltor derivative thereof. Therapeutic agent derivatives may betherapeutically active themselves or they may be prodrugs, which becomeactive upon further modification. Thus, in one embodiment, a therapeuticagent derivative retains some or all of the therapeutic activity ascompared to the unmodified agent, while in another embodiment, atherapeutic agent derivative is a prodrug that lacks therapeuticactivity, but becomes active upon further modification.

A. Nucleic Acids

In certain embodiments, lipid particles of the present invention areassociated with a nucleic acid, resulting in a nucleic acid-lipidparticle (e.g., LNP). In some embodiments, the nucleic acid is fullyencapsulated in the lipid particle. As used herein, the term “nucleicacid” includes any oligonucleotide or polynucleotide, with fragmentscontaining up to 60 nucleotides generally termed oligonucleotides, andlonger fragments termed polynucleotides. In particular embodiments,oligonucleotides of the invention are from about 15 to about 60nucleotides in length. Nucleic acid may be administered alone in thelipid particles of the invention, or in combination (e.g.,co-administered) with lipid particles of the invention comprisingpeptides, polypeptides, or small molecules such as conventional drugs.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally-occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also include polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake, reduced immunogenicity, and increasedstability in the presence of nucleases.

Oligonucleotides are generally classified as deoxyribooligonucleotidesor ribooligonucleotides. A deoxyribooligonucleotide consists of a5-carbon sugar called deoxyribose joined covalently to phosphate at the5′ and 3′ carbons of this sugar to form an alternating, unbranchedpolymer. A ribooligonucleotide consists of a similar repeating structurewhere the 5-carbon sugar is ribose.

The nucleic acid that is present in a lipid-nucleic acid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples ofdouble-stranded DNA are described herein and include, e.g., structuralgenes, genes including control and termination regions, andself-replicating systems such as viral or plasmid DNA. Examples ofdouble-stranded RNA are described herein and include, e.g., siRNA andother RNAi agents such as aiRNA and pre-miRNA. Single-stranded nucleicacids include, e.g., antisense oligonucleotides, ribozymes, maturemiRNA, and triplex-forming oligonucleotides.

Nucleic acids of the invention may be of various lengths, generallydependent upon the particular form of nucleic acid. For example, inparticular embodiments, plasmids or genes may be from about 1,000 toabout 100,000 nucleotide residues in length. In particular embodiments,oligonucleotides may range from about 10 to about 100 nucleotides inlength. In various related embodiments, oligonucleotides, bothsingle-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 60 nucleotides, from about 15 to about 60nucleotides, from about 20 to about 50 nucleotides, from about 15 toabout 30 nucleotides, or from about 20 to about 30 nucleotides inlength.

In particular embodiments, an oligonucleotide (or a strand thereof) ofthe invention specifically hybridizes to or is complementary to a targetpolynucleotide sequence. The terms “specifically hybridizable” and“complementary” as used herein indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. In some embodiments, anoligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target sequence interferes with the normalfunction of the target sequence to cause a loss of utility or expressiontherefrom, and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or, in the case of in vitro assays, under conditions in whichthe assays are conducted. Thus, the oligonucleotide may include 1, 2, 3,or more base substitutions as compared to the region of a gene or mRNAsequence that it is targeting or to which it specifically hybridizes.

1. siRNA

The siRNA component of the nucleic acid-lipid particles of the presentinvention is capable of silencing the expression of a target gene ofinterest. Each strand of the siRNA duplex is typically about 15 to about60 nucleotides in length, typically about 15 to about 30 nucleotides inlength. In certain embodiments, the siRNA comprises at least onemodified nucleotide. The modified siRNA is generally lessimmunostimulatory than a corresponding unmodified siRNA sequence andretains RNAi activity against the target gene of interest. In someembodiments, the modified siRNA contains at least one 2′OMe purine orpyrimidine nucleotide such as a 2′OMe-guanosine, 2′OMe-uridine,2′OMe-adenosine, and/or 2′OMe-cytosine nucleotide. In some embodiments,one or more of the uridine and/or guanosine nucleotides are modified.The modified nucleotides can be present in one strand (i.e., sense orantisense) or both strands of the siRNA. The siRNA sequences may haveoverhangs (e.g., 3′ or 5′ overhangs as described in Elbashir et al.,Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)), ormay lack overhangs (i.e., have blunt ends).

The modified siRNA generally comprises from about 1% to about 100%(e.g., about 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100%) modified nucleotides in the double-stranded region of thesiRNA duplex. In certain embodiments, one, two, three, four, five, six,seven, eight, nine, ten, or more of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides.

In some embodiments, less than about 25% (e.g., less than about 25%,24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides.

In other embodiments, from about 1% to about 25% (e.g., from about1%-25%, 2%-25%, 3%-25%, 4%-25%, 5%-25%, 6%-25%, 7%-25%, 8%-25%, 9%-25%,10%-25%, 11%-25%, 12%-25%, 13%-25%, 14%-25%, 15%-25%, 16%-25%, 17%-25%,18%-25%, 19%-25%, 20%-25%, 21%-25%, 22%-25%, 23%-25%, 24%-25%, etc.) orfrom about 1% to about 20% (e.g., from about 1%-20%, 2%-20%, 3%-20%,4%-20%, 5%-20%, 6%-20%, 7%-20%, 8%-20%, 9%-20%, 10%-20%, 11%-20%,12%-20%, 13%-20%, 14%-20%, 15%-20%, 16%-20%, 17%-20%, 18%-20%, 19%-20%,1%-19%, 2%-19%, 3%-19%, 4%-19%, 5%-19%, 6%-19%, 7%-19%, 8%-19%, 9%-19%,10%-19%, 11%-19%, 12%-19%, 13%-19%, 14%-19%, 15%-19%, 16%-19%, 17%-19%,18%-19%, 1%-18%, 2%-18%, 3%-18%, 4%-18%, 5%-18%, 6%-18%, 7%-18%, 8%-18%,9%-18%, 10%-18%, 11%-18%, 12%-18%, 13%-18%, 14%-18%, 15%-18%, 16%-18%,17%-18%, 1%-17%, 2%-17%, 3%-17%, 4%-17%, 5%-17%, 6%-17%, 7%-17%, 8%-17%,9%-17%, 10%-17%, 11%-17%, 12%-17%, 13%-17%, 14%-17%, 15%-17%, 16%-17%,1%-16%, 2%-16%, 3%-16%, 4%-16%, 5%-16%, 6%-16%, 7%-16%, 8%-16%, 9%-16%,10%-16%, 11%-16%, 12%-16%, 13%-16%, 14%-16%, 15%-16%, 1%-15%, 2%-15%,3%-15%, 4%-15%, 5%-15%, 6%-15%, 7%-15%, 8%-15%, 9%-15%, 10%-15%,11%-15%, 12%-15%, 13%-15%, 14%-15%, etc.) of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides.

In further embodiments, e.g., when one or both strands of the siRNA areselectively modified at uridine and/or guanosine nucleotides, theresulting modified siRNA can comprise less than about 30% modifiednucleotides (e.g., less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%,23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% modified nucleotides) or fromabout 1% to about 30% modified nucleotides (e.g., from about 1%-30%,2%-30%, 3%-30%, 4%-30%, 5%-30%, 6%-30%, 7%-30%, 8%-30%, 9%-30%, 10%-30%,11%-30%, 12%-30%, 13%-30%, 14%-30%, 15%-30%, 16%-30%, 17%-30%, 18%-30%,19%-30%, 20%-30%, 21%-30%, 22%-30%, 23%-30%, 24%-30%, 25%-30%, 26%-30%,27%-30%, 28%-30%, or 29%-30% modified nucleotides).

a. Selection of siRNA Sequences

Suitable siRNA sequences can be identified using any means known in theart. Typically, the methods described in Elbashir et al., Nature,411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) arecombined with rational design rules set forth in Reynolds et al., NatureBiotech., 22(3):326-330 (2004).

Generally, the nucleotide sequence 3′ of the AUG start codon of atranscript from the target gene of interest is scanned for dinucleotidesequences (e.g., AA, NA, CC, GG, or UU, wherein N=C, G, or U) (see,e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotidesimmediately 3′ to the dinucleotide sequences are identified as potentialsiRNA sequences (i.e., a target sequence or a sense strand sequence).Typically, the 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotidesimmediately 3′ to the dinucleotide sequences are identified as potentialsiRNA sequences. In some embodiments, the dinucleotide sequence is an AAor NA sequence and the 19 nucleotides immediately 3′ to the AA or NAdinucleotide are identified as potential siRNA sequences. siRNAsequences are usually spaced at different positions along the length ofthe target gene. To further enhance silencing efficiency of the siRNAsequences, potential siRNA sequences may be analyzed to identify sitesthat do not contain regions of homology to other coding sequences, e.g.,in the target cell or organism. For example, a suitable siRNA sequenceof about 21 base pairs typically will not have more than 16-17contiguous base pairs of homology to coding sequences in the target cellor organism. If the siRNA sequences are to be expressed from an RNA PolIII promoter, siRNA sequences lacking more than 4 contiguous A's or T'sare selected.

Once a potential siRNA sequence has been identified, a complementarysequence (i.e., an antisense strand sequence) can be designed. Apotential siRNA sequence can also be analyzed using a variety ofcriteria known in the art. For example, to enhance their silencingefficiency, the siRNA sequences may be analyzed by a rational designalgorithm to identify sequences that have one or more of the followingfeatures: (1) G/C content of about 25% to about 60% G/C; (2) at least 3A/Us at positions 15-19 of the sense strand; (3) no internal repeats;(4) an A at position 19 of the sense strand; (5) an A at position 3 ofthe sense strand; (6) a U at position 10 of the sense strand; (7) no G/Cat position 19 of the sense strand; and (8) no G at position 13 of thesense strand. siRNA design tools that incorporate algorithms that assignsuitable values of each of these features and are useful for selectionof siRNA can be found at, e.g., http://boz094.ust.hk/RNAi/siRNA. One ofskill in the art will appreciate that sequences with one or more of theforegoing characteristics may be selected for further analysis andtesting as potential siRNA sequences.

Additionally, potential siRNA sequences with one or more of thefollowing criteria can often be eliminated as siRNA: (1) sequencescomprising a stretch of 4 or more of the same base in a row; (2)sequences comprising homopolymers of Gs (i.e., to reduce possiblenon-specific effects due to structural characteristics of thesepolymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC,AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in arow; and (5) sequences comprising direct repeats of 4 or more baseswithin the candidates resulting in internal fold-back structures.However, one of skill in the art will appreciate that sequences with oneor more of the foregoing characteristics may still be selected forfurther analysis and testing as potential siRNA sequences.

In some embodiments, potential siRNA sequences may be further analyzedbased on siRNA duplex asymmetry as described in, e.g., Khvorova et al.,Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208 (2003).In other embodiments, potential siRNA sequences may be further analyzedbased on secondary structure at the target site as described in, e.g.,Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example,secondary structure at the target site can be modeled using the Mfoldalgorithm (available athttp://www.bioinfo.rpi.edu/applications/mfold/rna/form1.cgi) to selectsiRNA sequences which favor accessibility at the target site where lesssecondary structure in the form of base-pairing and stem-loops ispresent.

Once a potential siRNA sequence has been identified, the sequence can beanalyzed for the presence of any immunostimulatory properties, e.g.,using an in vitro cytokine assay or an in vivo animal model. Motifs inthe sense and/or antisense strand of the siRNA sequence such as GU-richmotifs (e.g., 5′-GU-3′,5′-UGU-3′,5′-GUGU-3′,5′-UGUGU-3′, etc.) can alsoprovide an indication of whether the sequence may be immunostimulatory.Once an siRNA molecule is found to be immunostimulatory, it can then bemodified to decrease its immunostimulatory properties as describedherein. As a non-limiting example, an siRNA sequence can be contactedwith a mammalian responder cell under conditions such that the cellproduces a detectable immune response to determine whether the siRNA isan immunostimulatory or a non-immunostimulatory siRNA. The mammalianresponder cell may be from a naïve mammal (i.e., a mammal that has notpreviously been in contact with the gene product of the siRNA sequence).The mammalian responder cell may be, e.g., a peripheral bloodmononuclear cell (PBMC), a macrophage, and the like. The detectableimmune response may comprise production of a cytokine or growth factorsuch as, e.g., TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-12, or a combinationthereof. An siRNA molecule identified as being immunostimulatory canthen be modified to decrease its immunostimulatory properties byreplacing at least one of the nucleotides on the sense and/or antisensestrand with modified nucleotides. For example, less than about 30%(e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) of thenucleotides in the double-stranded region of the siRNA duplex can bereplaced with modified nucleotides such as 2′OMe nucleotides. Themodified siRNA can then be contacted with a mammalian responder cell asdescribed above to confirm that its immunostimulatory properties havebeen reduced or abrogated.

Suitable in vitro assays for detecting an immune response include, butare not limited to, the double monoclonal antibody sandwich immunoassaytechnique of David et al. (U.S. Pat. No. 4,376,110);monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkhamand Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone,Edinburgh (1970)); the “Western blot” method of Gordon et al. (U.S. Pat.No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assays(ELISA) as described, for example, by Raines et al., J. Biol. Chem.,257:5154-5160 (1982); immunocytochemical techniques, including the useof fluorochromes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980));and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad.Sci. USA, 81:2396-2400 (1984)). In addition to the immunoassaysdescribed above, a number of other immunoassays are available, includingthose described in U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Thedisclosures of these references are herein incorporated by reference intheir entirety for all purposes.

A non-limiting example of an in vivo model for detecting an immuneresponse includes an in vivo mouse cytokine induction assay as describedin, e.g., Judge et al., Mol. Ther., 13:494-505 (2006). In certainembodiments, the assay that can be performed as follows: (1) siRNA canbe administered by standard intravenous injection in the lateral tailvein; (2) blood can be collected by cardiac puncture about 6 hours afteradministration and processed as plasma for cytokine analysis; and (3)cytokines can be quantified using sandwich ELISA kits according to themanufacturer's instructions (e.g., mouse and human IFN-α (PBLBiomedical; Piscataway, N.J.); human IL-6 and TNF-α (eBioscience; SanDiego, Calif.); and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; SanDiego, Calif.)).

Monoclonal antibodies that specifically bind cytokines and growthfactors are commercially available from multiple sources and can begenerated using methods known in the art (see, e.g., Kohler et al.,Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)).Generation of monoclonal antibodies has been previously described andcan be accomplished by any means known in the art (Buhring et al., inHybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, themonoclonal antibody is labeled (e.g., with any composition detectable byspectroscopic, photochemical, biochemical, electrical, optical, orchemical means) to facilitate detection.

b. Generating siRNA Molecules

siRNA can be provided in several forms including, e.g., as one or moreisolated small-interfering RNA (siRNA) duplexes, as longerdouble-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from atranscriptional cassette in a DNA plasmid. The siRNA sequences may haveoverhangs (e.g., 3′ or 5′ overhangs as described in Elbashir et al.,Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001), ormay lack overhangs (i.e., to have blunt ends).

An RNA population can be used to provide long precursor RNAs, or longprecursor RNAs that have substantial or complete identity to a selectedtarget sequence can be used to make the siRNA. The RNAs can be isolatedfrom cells or tissue, synthesized, and/or cloned according to methodswell known to those of skill in the art. The RNA can be a mixedpopulation (obtained from cells or tissue, transcribed from cDNA,subtracted, selected, etc.), or can represent a single target sequence.RNA can be naturally occurring (e.g., isolated from tissue or cellsamples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCRproducts or a cloned cDNA), or chemically synthesized.

To form a long dsRNA, for synthetic RNAs, the complement is alsotranscribed in vitro and hybridized to form a dsRNA. If a naturallyoccurring RNA population is used, the RNA complements are also provided(e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g.,by transcribing cDNAs corresponding to the RNA population, or by usingRNA polymerases. The precursor RNAs are then hybridized to form doublestranded RNAs for digestion. The dsRNAs can be directly administered toa subject or can be digested in vitro prior to administration.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see,U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Expressionlibraries are also well known to those of skill in the art. Additionalbasic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994). The disclosures of these references are herein incorporatedby reference in their entirety for all purposes.

Typically, siRNA are chemically synthesized. The oligonucleotides thatcomprise the siRNA molecules of the invention can be synthesized usingany of a variety of techniques known in the art, such as those describedin Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al.,Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res.,23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59(1997). The synthesis of oligonucleotides makes use of common nucleicacid protecting and coupling groups, such as dimethoxytrityl at the5′-end and phosphoramidites at the 3′-end. As a non-limiting example,small scale syntheses can be conducted on an Applied Biosystemssynthesizer using a 0.2 μmol scale protocol. Alternatively, syntheses atthe 0.2 μmol scale can be performed on a 96-well plate synthesizer fromProtogene (Palo Alto, Calif.). However, a larger or smaller scale ofsynthesis is also within the scope of this invention. Suitable reagentsfor oligonucleotide synthesis, methods for RNA deprotection, and methodsfor RNA purification are known to those of skill in the art.

siRNA molecules can also be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousoligonucleotide fragment or strand separated by a cleavable linker thatis subsequently cleaved to provide separate fragments or strands thathybridize to form the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker. The tandem synthesis of siRNA can bereadily adapted to both multiwell/multiplate synthesis platforms as wellas large scale synthesis platforms employing batch reactors, synthesiscolumns, and the like. Alternatively, siRNA molecules can be assembledfrom two distinct oligonucleotides, wherein one oligonucleotidecomprises the sense strand and the other comprises the antisense strandof the siRNA. For example, each strand can be synthesized separately andjoined together by hybridization or ligation following synthesis and/ordeprotection. In certain other instances, siRNA molecules can besynthesized as a single continuous oligonucleotide fragment, where theself-complementary sense and antisense regions hybridize to form ansiRNA duplex having hairpin secondary structure.

c. Modifying siRNA Sequences

In certain aspects, siRNA molecules comprise a duplex having two strandsand at least one modified nucleotide in the double-stranded region,wherein each strand is about 15 to about 60 nucleotides in length.Advantageously, the modified siRNA is less immunostimulatory than acorresponding unmodified siRNA sequence, but retains the capability ofsilencing the expression of a target sequence. In some embodiments, thedegree of chemical modifications introduced into the siRNA moleculestrikes a balance between reduction or abrogation of theimmunostimulatory properties of the siRNA and retention of RNAiactivity. As a non-limiting example, an siRNA molecule that targets agene of interest can be minimally modified (e.g., less than about 30%,25%, 20%, 15%, 10%, or 5% modified) at selective uridine and/orguanosine nucleotides within the siRNA duplex to eliminate the immuneresponse generated by the siRNA while retaining its capability tosilence target gene expression.

Examples of modified nucleotides suitable for use in the inventioninclude, but are not limited to, ribonucleotides having a 2′-O-methyl(2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl,2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.Modified nucleotides having a Northern conformation such as thosedescribed in, e.g., Saenger, Principles of Nucleic Acid Structure,Springer-Verlag Ed. (1984), are also suitable for use in siRNAmolecules. Such modified nucleotides include, without limitation, lockednucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl)(MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro (2′Cl) nucleotides, and 2′-azidonucleotides. In certain instances, the siRNA molecules described hereininclude one or more G-clamp nucleotides. A G-clamp nucleotide refers toa modified cytosine analog wherein the modifications confer the abilityto hydrogen bond both Watson-Crick and Hoogsteen faces of acomplementary guanine nucleotide within a duplex (see, e.g., Lin et al.,J. Am. Chem. Soc., 120:8531-8532 (1998)). In addition, nucleotideshaving a nucleotide base analog such as, for example, C-phenyl,C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, andnitroazole derivatives such as 3-nitropyrrole, 4-nitroindole,5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res.,29:2437-2447 (2001)) can be incorporated into siRNA molecules.

In certain embodiments, siRNA molecules may further comprise one or morechemical modifications such as terminal cap moieties, phosphate backbonemodifications, and the like. Examples of terminal cap moieties include,without limitation, inverted deoxy abasic residues, glycerylmodifications, 4′,5′-methylene nucleotides, 1-(β-D-erythrofuranosyl)nucleotides, 4′-thio nucleotides, carbocyclic nucleotides,1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modifiedbase nucleotides, threo-pentofuranosyl nucleotides, acyclic 3′,4′-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties,3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties,3′-2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties,5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties,5′-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropylphosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate,hydroxypropyl phosphate, 1,4-butanediol phosphate, 3′-phosphoramidate,5′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate,and bridging or non-bridging methylphosphonate or 5′-mercapto moieties(see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron49:1925 (1993)). Non-limiting examples of phosphate backbonemodifications (i.e., resulting in modified internucleotide linkages)include phosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate, carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker etal., Nucleic Acid Analogues: Synthesis and Properties, in ModernSynthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39 (1994)). Such chemical modifications canoccur at the 5′-end and/or 3′-end of the sense strand, antisense strand,or both strands of the siRNA. The disclosures of these references areherein incorporated by reference in their entirety for all purposes.

In some embodiments, the sense and/or antisense strand of the siRNAmolecule can further comprise a 3′-terminal overhang having about 1 toabout 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides and/or anycombination of modified and unmodified nucleotides. Additional examplesof modified nucleotides and types of chemical modifications that can beintroduced into siRNA molecules are described, e.g., in UK Patent No. GB2,397,818 B and U.S. Patent Publication Nos. 20040192626, 20050282188,and 20070135372, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

The siRNA molecules described herein can optionally comprise one or morenon-nucleotides in one or both strands of the siRNA. As used herein, theterm “non-nucleotide” refers to any group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including sugar and/or phosphate substitutions, andallows the remaining bases to exhibit their activity. The group orcompound is abasic in that it does not contain a commonly recognizednucleotide base such as adenosine, guanine, cytosine, uracil, or thymineand therefore lacks a base at the 1′-position.

In other embodiments, chemical modification of the siRNA comprisesattaching a conjugate to the siRNA molecule. The conjugate can beattached at the 5′ and/or 3′-end of the sense and/or antisense strand ofthe siRNA via a covalent attachment such as, e.g., a biodegradablelinker. The conjugate can also be attached to the siRNA, e.g., through acarbamate group or other linking group (see, e.g., U.S. PatentPublication Nos. 20050074771, 20050043219, and 20050158727). In certaininstances, the conjugate is a molecule that facilitates the delivery ofthe siRNA into a cell. Examples of conjugate molecules suitable forattachment to siRNA include, without limitation, steroids such ascholesterol, glycols such as polyethylene glycol (PEG), human serumalbumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates(e.g., folic acid, folate analogs and derivatives thereof), sugars(e.g., galactose, galactosamine, N-acetyl galactosamine, glucose,mannose, fructose, fucose, etc.), phospholipids, peptides, ligands forcellular receptors capable of mediating cellular uptake, andcombinations thereof (see, e.g., U.S. Patent Publication Nos.20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423).Other examples include the lipophilic moiety, vitamin, polymer, peptide,protein, nucleic acid, small molecule, oligosaccharide, carbohydratecluster, intercalator, minor groove binder, cleaving agent, andcross-linking agent conjugate molecules described in U.S. PatentPublication Nos. 20050119470 and 20050107325. Yet other examples includethe 2′-O-alkyl amine, 2′-β-alkoxyalkyl amine, polyamine, C5-cationicmodified pyrimidine, cationic peptide, guanidinium group, amidininiumgroup, cationic amino acid conjugate molecules described in U.S. PatentPublication No. 20050153337. Additional examples include the hydrophobicgroup, membrane active compound, cell penetrating compound, celltargeting signal, interaction modifier, and steric stabilizer conjugatemolecules described in U.S. Patent Publication No. 20040167090. Furtherexamples include the conjugate molecules described in U.S. PatentPublication No. 20050239739. The type of conjugate used and the extentof conjugation to the siRNA molecule can be evaluated for improvedpharmacokinetic profiles, bioavailability, and/or stability of the siRNAwhile retaining RNAi activity. As such, one skilled in the art canscreen siRNA molecules having various conjugates attached thereto toidentify ones having improved properties and full RNAi activity usingany of a variety of well-known in vitro cell culture or in vivo animalmodels. The disclosures of the above-described patent documents areherein incorporated by reference in their entirety for all purposes.

d. Target Genes

In certain embodiments, the nucleic acid component (e.g., siRNA) of thenucleic acid-lipid particles described herein can be used todownregulate or silence the translation (i.e., expression) of a gene ofinterest. Genes of interest include, but are not limited to, genesassociated with viral infection and survival, genes associated withmetabolic diseases and disorders (e.g., liver diseases and disorders),genes associated with tumorigenesis and cell transformation (e.g.,cancer), angiogenic genes, immunomodulator genes such as thoseassociated with inflammatory and autoimmune responses, ligand receptorgenes, and genes associated with neurodegenerative disorders. In certainembodiments, the gene of interest is expressed in hepatocytes.

Genes associated with viral infection and survival include thoseexpressed by a virus in order to bind, enter, and replicate in a cell.Of particular interest are viral sequences associated with chronic viraldiseases. Viral sequences of particular interest include sequences ofFiloviruses such as Ebola virus and Marburg virus (see, e.g., Geisbertet al., J. Infect. Dis., 193:1650-1657 (2006)); Arenaviruses such asLassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabiavirus (Buchmeier et al., Arenaviridae: the viruses and theirreplication, In: FIELDS VIROLOGY, Knipe et al. (eds.), 4th ed.,Lippincott-Raven, Philadelphia, (2001)); Influenza viruses such asInfluenza A, B, and C viruses, (see, e.g., Steinhauer et al., Annu RevGenet., 36:305-332 (2002); and Neumann et al., J Gen Virol.,83:2635-2662 (2002)); Hepatitis viruses (see, e.g., Hamasaki et al.,FEBS Lett., 543:51 (2003); Yokota et al., EMBO Rep., 4:602 (2003);Schlomai et al., Hepatology, 37:764 (2003); Wilson et al., Proc. Natl.Acad. Sci. USA, 100:2783 (2003); Kapadia et al., Proc. Natl. Acad. Sci.USA, 100:2014 (2003); and FIELDS VIROLOGY, Knipe et al. (eds.), 4th ed.,Lippincott-Raven, Philadelphia (2001)); Human Immunodeficiency Virus(HIV) (Banerjea et al., Mol. Ther., 8:62 (2003); Song et al., J. Virol.,77:7174 (2003); Stephenson, JAMA, 289:1494 (2003); Qin et al., Proc.Natl. Acad. Sci. USA, 100:183 (2003)); Herpes viruses (Jia et al., J.Virol., 77:3301 (2003)); and Human Papilloma Viruses (HPV) (Hall et al.,J. Virol., 77:6066 (2003); Jiang et al., Oncogene, 21:6041 (2002)).

Exemplary Filovirus nucleic acid sequences that can be silenced include,but are not limited to, nucleic acid sequences encoding structuralproteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase protein(L-pol)) and membrane-associated proteins (e.g., VP40, glycoprotein(GP), VP24). Complete genome sequences for Ebola virus are set forth in,e.g., Genbank Accession Nos. NC_002549; AY769362; NC_006432; NC_004161;AY729654; AY354458; AY142960; AB050936; AF522874; AF499101; AF272001;and AF086833. Ebola virus VP24 sequences are set forth in, e.g., GenbankAccession Nos. U77385 and AY058897. Ebola virus L-pol sequences are setforth in, e.g., Genbank Accession No. X67110. Ebola virus VP40 sequencesare set forth in, e.g., Genbank Accession No. AY058896. Ebola virus NPsequences are set forth in, e.g., Genbank Accession No. AY058895. Ebolavirus GP sequences are set forth in, e.g., Genbank Accession No.AY058898; Sanchez et al., Virus Res., 29:215-240 (1993); Will et al., J.Virol., 67:1203-1210 (1993); Volchkov et al., FEBS Lett., 305:181-184(1992); and U.S. Pat. No. 6,713,069. Additional Ebola virus sequencesare set forth in, e.g., Genbank Accession Nos. L11365 and X61274.Complete genome sequences for Marburg virus are set forth in, e.g.,Genbank Accession Nos. NC_001608; AY430365; AY430366; and AY358025.Marburg virus GP sequences are set forth in, e.g., Genbank AccessionNos. AF005734; AF005733; and AF005732. Marburg virus VP35 sequences areset forth in, e.g., Genbank Accession Nos. AF005731 and AF005730.Additional Marburg virus sequences are set forth in, e.g., GenbankAccession Nos. X64406; Z29337; AF005735; and Z12132. Non-limitingexamples of siRNA molecules targeting Ebola virus and Marburg virusnucleic acid sequences include those described in U.S. PatentPublication No. 20070135370, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

Exemplary Influenza virus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences encodingnucleoprotein (NP), matrix proteins (M1 and M2), nonstructural proteins(NS1 and NS2), RNA polymerase (PA, PB1, PB2), neuraminidase (NA), andhaemagglutinin (HA). Influenza A NP sequences are set forth in, e.g.,Genbank Accession Nos. NC_004522; AY818138; AB166863; AB188817;AB189046; AB189054; AB189062; AY646169; AY646177; AY651486; AY651493;AY651494; AY651495; AY651496; AY651497; AY651498; AY651499; AY651500;AY651501; AY651502; AY651503; AY651504; AY651505; AY651506; AY651507;AY651509; AY651528; AY770996; AY790308; AY818138; and AY818140.Influenza A PA sequences are set forth in, e.g., Genbank Accession Nos.AY818132; AY790280; AY646171; AY818132; AY818133; AY646179; AY818134;AY551934; AY651613; AY651610; AY651620; AY651617; AY651600; AY651611;AY651606; AY651618; AY651608; AY651607; AY651605; AY651609; AY651615;AY651616; AY651640; AY651614; AY651612; AY651621; AY651619; AY770995;and AY724786. Non-limiting examples of siRNA molecules targetingInfluenza virus nucleic acid sequences include those described in U.S.Patent Publication No. 20070218122, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

Exemplary hepatitis virus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences involved intranscription and translation (e.g., En1, En2, X, P) and nucleic acidsequences encoding structural proteins (e.g., core proteins including Cand C-related proteins, capsid and envelope proteins including S, M,and/or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY,supra). Exemplary Hepatitis C virus (HCV) nucleic acid sequences thatcan be silenced include, but are not limited to, the 5′-untranslatedregion (5′-UTR), the 3′-untranslated region (3′-UTR), the polyproteintranslation initiation codon region, the internal ribosome entry site(IRES) sequence, and/or nucleic acid sequences encoding the coreprotein, the E1 protein, the E2 protein, the p7 protein, the NS2protein, the NS3 protease/helicase, the NS4A protein, the NS4B protein,the NS5A protein, and/or the NS5B RNA-dependent RNA polymerase. HCVgenome sequences are set forth in, e.g., Genbank Accession Nos.NC_004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b), NC_009823 (HCVgenotype 2), NC_009824 (HCV genotype 3), NC_009825 (HCV genotype 4),NC_009826 (HCV genotype 5), and NC_009827 (HCV genotype 6). Hepatitis Avirus nucleic acid sequences are set forth in, e.g., Genbank AccessionNo. NC_001489; Hepatitis B virus nucleic acid sequences are set forthin, e.g., Genbank Accession No. NC_003977; Hepatitis D virus nucleicacid sequence are set forth in, e.g., Genbank Accession No. NC_001653;Hepatitis E virus nucleic acid sequences are set forth in, e.g., GenbankAccession No. NC_001434; and Hepatitis G virus nucleic acid sequencesare set forth in, e.g., Genbank Accession No. NC_001710. Silencing ofsequences that encode genes associated with viral infection and survivalcan conveniently be used in combination with the administration ofconventional agents used to treat the viral condition. Non-limitingexamples of siRNA molecules targeting hepatitis virus nucleic acidsequences include those described in U.S. Patent Publication Nos.20060281175, 20050058982, and 20070149470; U.S. Pat. No. 7,348,314; andU.S. Provisional Application No. 61/162,127, filed Mar. 20, 2009, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

Genes associated with metabolic diseases and disorders (e.g., disordersin which the liver is the target and liver diseases and disorders)include, for example, genes expressed in dyslipidemia (e.g., liver Xreceptors such as LXRα and LXRβ (Genback Accession No. NM_007121),farnesoid X receptors (FXR) (Genbank Accession No. NM_005123),sterol-regulatory element binding protein (SREBP), site-1 protease(SIP), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG coenzyme-Areductase), apolipoprotein B (ApoB) (Genbank Accession No. NM_000384),apolipoprotein CIII (ApoC3) (Genbank Accession Nos. NM_000040 andNG_008949 REGION: 5001.8164), and apolipoprotein E (ApoE) (GenbankAccession Nos. NM_000041 and NG_007084 REGION: 5001.8612)); and diabetes(e.g., glucose 6-phosphatase) (see, e.g., Forman et al., Cell, 81:687(1995); Seol et al., Mol. Endocrinol., 9:72 (1995), Zavacki et al.,Proc. Natl. Acad. Sci. USA, 94:7909 (1997); Sakai et al., Cell,85:1037-1046 (1996); Duncan et al., J. Biol. Chem., 272:12778-12785(1997); Willy et al., Genes Dev., 9:1033-1045 (1995); Lehmann et al., J.Biol. Chem., 272:3137-3140 (1997); Janowski et al., Nature, 383:728-731(1996); and Peet et al., Cell, 93:693-704 (1998)). One of skill in theart will appreciate that genes associated with metabolic diseases anddisorders (e.g., diseases and disorders in which the liver is a targetand liver diseases and disorders) include genes that are expressed inthe liver itself as well as and genes expressed in other organs andtissues. Silencing of sequences that encode genes associated withmetabolic diseases and disorders can conveniently be used in combinationwith the administration of conventional agents used to treat the diseaseor disorder. Non-limiting examples of siRNA molecules targeting the ApoBgene include those described in U.S. Patent Publication No. 20060134189,the disclosure of which is herein incorporated by reference in itsentirety for all purposes. Non-limiting examples of siRNA moleculestargeting the ApoC3 gene include those described in U.S. ProvisionalApplication No. 61/147,235, filed Jan. 26, 2009, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

Examples of gene sequences associated with tumorigenesis and celltransformation (e.g., cancer or other neoplasia) include mitotickinesins such as Eg5 (KSP, KIF11; Genbank Accession No. NM_004523);serine/threonine kinases such as polo-like kinase 1 (PLK-1) (GenbankAccession No. NM_005030; Barr et al., Nat. Rev. Mol. Cell. Biol.,5:429-440 (2004)); tyrosine kinases such as WEEl (Genbank Accession Nos.NM_003390 and NM_001143976); inhibitors of apoptosis such as XIAP(Genbank Accession No. NM_001167); COP9 signalosome subunits such asCSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank Accession No. NM_006837);CSN6, CSN7A, CSN7B, and CSN8; ubiquitin ligases such as COP1 (RFWD2;Genbank Accession Nos. NM_022457 and NM_001001740); and histonedeacetylases such as HDAC1, HDAC2 (Genbank Accession No. NM_001527),HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, etc. Non-limitingexamples of siRNA molecules targeting the Eg5 and XIAP genes includethose described in U.S. patent application Ser. No. 11/807,872, filedMay 29, 2007, the disclosure of which is herein incorporated byreference in its entirety for all purposes. Non-limiting examples ofsiRNA molecules targeting the PLK-1 gene include those described in U.S.Patent Publication Nos. 20050107316 and 20070265438; and U.S. patentapplication Ser. No. 12/343,342, filed Dec. 23, 2008, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. Non-limiting examples of siRNA molecules targeting the CSN5gene include those described in U.S. Provisional Application No.61/045,251, filed Apr. 15, 2008, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

Additional examples of gene sequences associated with tumorigenesis andcell transformation include translocation sequences such as MLL fusiongenes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al.,Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2,AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003));overexpressed sequences such as multidrug resistance genes (Nieth etal., FEBS Lett., 545:144 (2003); Wu et al, Cancer Res. 63:1515 (2003)),cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et al., Genes Dev.,16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291(2003)), telomerase genes (Kosciolek et al., Mol Cancer Ther., 2:209(2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR/ErbB1(Genbank Accession Nos. NM_005228, NM_201282, NM_201283, and NM_201284;see also, Nagy et al. Exp. Cell Res., 285:39-49 (2003), ErbB2/HER-2(Genbank Accession Nos. NM_004448 and NM_001005862), ErbB3 (GenbankAccession Nos. NM_001982 and NM_001005915), and ErbB4 (Genbank AccessionNos. NM_005235 and NM_001042599); and mutated sequences such as RAS(reviewed in Tuschl and Borkhardt, Mol. Interventions, 2:158 (2002)).Non-limiting examples of siRNA molecules targeting the EGFR gene includethose described in U.S. patent application Ser. No. 11/807,872, filedMay 29, 2007, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

Silencing of sequences that encode DNA repair enzymes find use incombination with the administration of chemotherapeutic agents (Colliset al., Cancer Res., 63:1550 (2003)). Genes encoding proteins associatedwith tumor migration are also target sequences of interest, for example,integrins, selectins, and metalloproteinases. The foregoing examples arenot exclusive. Those of skill in the art will understand that any wholeor partial gene sequence that facilitates or promotes tumorigenesis orcell transformation, tumor growth, or tumor migration can be included asa template sequence.

Angiogenic genes are able to promote the formation of new vessels. Ofparticular interest is vascular endothelial growth factor (VEGF) (Reichet al., Mol. Vis., 9:210 (2003)) or VEGFR. siRNA sequences that targetVEGFR are set forth in, e.g., GB 2396864; U.S. Patent Publication No.20040142895; and CA 2456444, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

Anti-angiogenic genes are able to inhibit neovascularization. Thesegenes are particularly useful for treating those cancers in whichangiogenesis plays a role in the pathological development of thedisease. Examples of anti-angiogenic genes include, but are not limitedto, endostatin (see, e.g., U.S. Pat. No. 6,174,861), angiostatin (see,e.g., U U.S. Pat. No. 5,639,725), and VEGFR2 (see, e.g., Decaussin etal., J. Pathol., 188: 369-377 (1999)), the disclosures of which areherein incorporated by reference in their entirety for all purposes.

Immunomodulator genes are genes that modulate one or more immuneresponses. Examples of immunomodulator genes include, withoutlimitation, cytokines such as growth factors (e.g., TGF-α, TGF-β, EGF,FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2,IL-4, IL-12 (Hill et al., J. Immunol., 171:691 (2003)), IL-15, IL-18,IL-20, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.) and TNF. Fasand Fas ligand genes are also immunomodulator target sequences ofinterest (Song et al., Nat. Med., 9:347 (2003)). Genes encodingsecondary signaling molecules in hematopoietic and lymphoid cells arealso included in the present invention, for example, Tec family kinasessuch as Bruton's tyrosine kinase (Btk) (Heinonen et al., FEBS Lett.,527:274 (2002)).

Cell receptor ligands include ligands that are able to bind to cellsurface receptors (e.g., insulin receptor, EPO receptor, G-proteincoupled receptors, receptors with tyrosine kinase activity, cytokinereceptors, growth factor receptors, etc.), to modulate (e.g., inhibit,activate, etc.) the physiological pathway that the receptor is involvedin (e.g., glucose level modulation, blood cell development, mitogenesis,etc.). Examples of cell receptor ligands include, but are not limitedto, cytokines, growth factors, interleukins, interferons, erythropoietin(EPO), insulin, glucagon, G-protein coupled receptor ligands, etc.Templates coding for an expansion of trinucleotide repeats (e.g., CAGrepeats) find use in silencing pathogenic sequences in neurodegenerativedisorders caused by the expansion of trinucleotide repeats, such asspinobulbular muscular atrophy and Huntington's Disease (Caplen et al.,Hum. Mol. Genet., 11:175 (2002)).

Certain other target genes, which may be targeted by a nucleic acid(e.g., by siRNA) to downregulate or silence the expression of the gene,include but are not limited to, Actin, Alpha 2, Smooth Muscle, Aorta(ACTA2), Alcohol dehydrogenase 1A (ADH1A), Alcohol dehydrogenase 4(ADH4), Alcohol dehydrogenase 6 (ADH6), Afamin (AFM), Angiotensinogen(AGT), Serine-pyruvate aminotransferase (AGXT), Alpha-2-HS-glycoprotein(AHSG), Aldo-keto reductase family 1 member C4 (AKR1C4), Serum albumin(ALB), alpha-1-microglobulin/bikunin precursor (AMBP),Angiopoietin-related protein 3 (ANGPTL3), Serum amyloid P-component(APCS), Apolipoprotein A-II (APOA2), Apolipoprotein B-100 (APOB),Apolipoprotein C3 (APOC3), Apolipoprotein C-IV (APOC4), Apolipoprotein F(APOF), Beta-2-glycoprotein 1 (APOH), Aquaporin-9 (AQP9), Bileacid-CoA:amino acid N-acyltransferase (BAAT), C4b-binding protein betachain (C4BPB), Putative uncharacterized protein encoded by LINC01554(C5orf27), Complement factor 3 (C3), Complement Factor 5 (C5),Complement component C6 (C6), Complement component C8 alpha chain (C8A),Complement component C8 beta chain (C8B), Complement component C8 gammachain (C8G), Complement component C9 (C9), Calmodulin BindingTranscription Activator 1 (CAMTA1), CD38 (CD38), Complement Factor B(CFB), Complement factor H-related protein 1 (CFHR1), Complement factorH-related protein 2 (CFHR2), Complement factor H-related protein 3(CFHR3), Cannabinoid receptor 1 (CNR1), ceruloplasmin (CP),carboxypeptidase B2 (CPB2), Connective tissue growth factor (CTGF),C-X-C motif chemokine 2 (CXCL2), Cytochrome P450 1A2 (CYP1A2),Cytochrome P450 2A6 (CYP2A6), Cytochrome P450 2C8 (CYP2C8), CytochromeP450 2C9 (CYP2C9), Cytochrome P450 Family 2 Subfamily D Member 6(CYP2D6), Cytochrome P450 2E1 (CYP2E1), Phylloquinone omega-hydroxylaseCYP4F2 (CYP4F2), 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase(CYP8B1), Dipeptidyl peptidase 4 (DPP4), coagulation factor 12 (F12),coagulation factor II (thrombin) (F2), coagulation factor IX (F9),fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), fibrinogengamma chain (FGG), fibrinogen-like 1 (FGL1), flavin containingmonooxygenase 3 (FMO3), flavin containing monooxygenase 5 (FMO5),group-specific component (vitamin D binding protein) (GC), Growthhormone receptor (GHR), glycine N-methyltransferase (GNMT), hyaluronanbinding protein 2 (HABP2), hepcidin antimicrobial peptide (HAMP),hydroxyacid oxidase (glycolate oxidase) 1 (HAO1), HGF activator (HGFAC),haptoglobin-related protein; haptoglobin (HPR), hemopexin (HPX),histidine-rich glycoprotein (HRG), hydroxysteroid (11-beta)dehydrogenase 1 (HSD11B1), hydroxysteroid (17-beta) dehydrogenase 13(HSD17B13), Inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1),Inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2),Inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3),Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Prekallikrein(KLKB1), Lactate dehydrogenase A (LDHA), liver expressed antimicrobialpeptide 2 (LEAP2), leukocyte cell-derived chemotaxin 2 (LECT2),Lipoprotein (a) (LPA), mannan-binding lectin serine peptidase 2 (MASP2),S-adenosylmethionine synthase isoform type-1 (MAT1A), NADPH Oxidase 4(NOX4), Poly [ADP-ribose] polymerase 1 (PARP1), paraoxonase 1 (PON1),paraoxonase 3 (PON3), Vitamin K-dependent protein C (PROC), Retinoldehydrogenase 16 (RDH16), serum amyloid A4, constitutive (SAA4), serinedehydratase (SDS), Serpin Family A Member 1 (SERPINA1), Serpin A11(SERPINA11), Kallistatin (SERPINA4), Corticosteroid-binding globulin(SERPINA6), Antithrombin-III (SERPINC1), Heparin cofactor 2 (SERPIND1),Serpin Family H Member 1 (SERPINH1), Solute Carrier Family 5 Member 2(SLC5A2), Sodium/bile acid cotransporter (SLC10A1), Solute carrierfamily 13 member 5 (SLC13A5), Solute carrier family 22 member 1(SLC22A1), Solute carrier family 25 member 47 (SLC25A47), Solute carrierfamily 2, facilitated glucose transporter member 2 (SLC2A2),Sodium-coupled neutral amino acid transporter 4 (SLC38A4), Solutecarrier organic anion transporter family member 1B1 (SLCO1B1),Sphingomyelin Phosphodiesterase 1 (SMPD1), Bile salt sulfotransferase(SULT2A1), tyrosine aminotransferase (TAT), tryptophan 2,3-dioxygenase(TDO2), UDP glucuronosyltransferase 2 family, polypeptide B10 (UGT2B10),UDP glucuronosyltransferase 2 family, polypeptide B15 (UGT2B15), UDPglucuronosyltransferase 2 family, polypeptide B4 (UGT2B4) andvitronectin (VTN).

In addition to its utility in silencing the expression of any of theabove-described genes for therapeutic purposes, certain nucleic acids(e.g., siRNA) described herein are also useful in research anddevelopment applications as well as diagnostic, prophylactic,prognostic, clinical, and other healthcare applications. As anon-limiting example, certain nucleic acids (e.g., siRNA) can be used intarget validation studies directed at testing whether a gene of interesthas the potential to be a therapeutic target. Certain nucleic acids(e.g., siRNA) can also be used in target identification studies aimed atdiscovering genes as potential therapeutic targets.

2. CRISPR

Targeted genome editing has progressed from being a niche technology toa method used by many biological researchers. This progression has beenlargely fueled by the emergence of the clustered, regularly interspaced,short palindromic repeat (CRISPR) technology (see, e.g., Sander et al.,Nature Biotechnology, 32(4), 347-355, including SupplementaryInformation (2014) and International Publication Numbers WO 2016/197132and WO 2016/197133). Accordingly, provided herein are improvements(e.g., lipid nanoparticles and formulations thereof) that can be used incombination with CRISPR technology to treat diseases, such as HBV.Regarding the targets for using CRISPR, the guide RNA (gRNA) utilized inthe CRISPR technology can be designed to target specifically identifiedsequences, e.g., target genes, e.g., of the HBV genome. Examples of suchtarget sequences are provided in International Publication Number WO2016/197132. Further, International Publication Number WO 2013/151665(e.g., see Table 6; which document is specifically incorporated byreference, particularly including Table 6, and the associated SequenceListing) describes about 35,000 mRNA sequences, claimed in the contextof an mRNA expression construct. Certain embodiments of the presentinvention utilize CRISPR technology to target the expression of any ofthese sequences. Certain embodiments of the present invention may alsoutilize CRISPR technology to target the expression of a target genediscussed herein.

3. aiRNA

Like siRNA, asymmetrical interfering RNA (aiRNA) can recruit theRNA-induced silencing complex (RISC) and lead to effective silencing ofa variety of genes in mammalian cells by mediating sequence-specificcleavage of the target sequence between nucleotide 10 and 11 relative tothe 5′ end of the antisense strand (Sun et al., Nat. Biotech.,26:1379-1382 (2008)). Typically, an aiRNA molecule comprises a short RNAduplex having a sense strand and an antisense strand, wherein the duplexcontains overhangs at the 3′ and 5′ ends of the antisense strand. TheaiRNA is generally asymmetric because the sense strand is shorter onboth ends when compared to the complementary antisense strand. In someaspects, aiRNA molecules may be designed, synthesized, and annealedunder conditions similar to those used for siRNA molecules. As anon-limiting example, aiRNA sequences may be selected and generatedusing the methods described above for selecting siRNA sequences.

In another embodiment, aiRNA duplexes of various lengths (e.g., about10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more typically12, 13, 14, 15, 16, 17, 18, 19, or base pairs) may be designed withoverhangs at the 3′ and 5′ ends of the antisense strand to target anmRNA of interest. In certain instances, the sense strand of the aiRNAmolecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or20 nucleotides in length. In certain other instances, the antisensestrand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and is typically about 20-24, 21-22, or 21-23 nucleotides inlength.

In some embodiments, the 5′ antisense overhang contains one, two, three,four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”, etc.).In other embodiments, the 3′ antisense overhang contains one, two,three, four, or more nontargeting nucleotides (e.g., “AA”, “IJU”,“dTdT”, etc.). In certain aspects, the aiRNA molecules described hereinmay comprise one or more modified nucleotides, e.g., in thedouble-stranded (duplex) region and/or in the antisense overhangs. As anon-limiting example, aiRNA sequences may comprise one or more of themodified nucleotides described above for siRNA sequences. In oneembodiment, the aiRNA molecule comprises 2′OMe nucleotides such as, forexample, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, ormixtures thereof.

In certain embodiments, aiRNA molecules may comprise an antisense strandwhich corresponds to the antisense strand of an siRNA molecule, e.g.,one of the siRNA molecules described herein. In other embodiments, aiRNAmolecules may be used to silence the expression of any of the targetgenes set forth above, such as, e.g., genes associated with viralinfection and survival, genes associated with metabolic diseases anddisorders, genes associated with tumorigenesis and cell transformation,angiogenic genes, immunomodulator genes such as those associated withinflammatory and autoimmune responses, ligand receptor genes, and genesassociated with neurodegenerative disorders.

4. miRNA

Generally, microRNAs (miRNA) are single-stranded RNA molecules of about21-23 nucleotides in length which regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed, but miRNAs are nottranslated into protein (non-coding RNA); instead, each primarytranscript (a pri-miRNA) is processed into a short stem-loop structurecalled a pre-miRNA and finally into a functional mature miRNA. MaturemiRNA molecules are either partially or completely complementary to oneor more messenger RNA (mRNA) molecules, and their main function is todownregulate gene expression. The identification of miRNA molecules isdescribed, e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau etal., Science, 294:858-862; and Lee et al., Science, 294:862-864.

The genes encoding miRNA are much longer than the processed mature miRNAmolecule. miRNA are first transcribed as primary transcripts orpri-miRNA with a cap and poly-A tail and processed to short,^(˜)70-nucleotide stem-loop structures known as pre-miRNA in the cellnucleus. This processing is performed in animals by a protein complexknown as the Microprocessor complex, consisting of the nuclease Droshaand the double-stranded RNA binding protein Pasha (Denli et al., Nature,432:231-235 (2004)). These pre-miRNA are then processed to mature miRNAin the cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC)(Bernstein et al., Nature, 409:363-366 (2001). Either the sense strandor antisense strand of DNA can function as templates to give rise tomiRNA.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC complex.This strand is known as the guide strand and is selected by theargonaute protein, the catalytically active RNase in the RISC complex,on the basis of the stability of the 5′ end (Preall et al., Curr. Biol.,16:530-535 (2006)). The remaining strand, known as the anti-guide orpassenger strand, is degraded as a RISC complex substrate (Gregory etal., Cell, 123:631-640 (2005)). After integration into the active RISCcomplex, miRNAs base pair with their complementary mRNA molecules andinduce target mRNA degradation and/or translational silencing.

Mammalian miRNA molecules are usually complementary to a site in the 3′UTR of the target mRNA sequence. In certain instances, the annealing ofthe miRNA to the target mRNA inhibits protein translation by blockingthe protein translation machinery. In certain other instances, theannealing of the miRNA to the target mRNA facilitates the cleavage anddegradation of the target mRNA through a process similar to RNAinterference (RNAi). miRNA may also target methylation of genomic siteswhich correspond to targeted mRNA. Generally, miRNA function inassociation with a complement of proteins collectively termed the miRNP.

In certain aspects, the miRNA molecules described herein are about15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and are typically about 20-24, 21-22, or 21-23 nucleotides inlength. In certain other aspects, miRNA molecules may comprise one ormore modified nucleotides. As a non-limiting example, miRNA sequencesmay comprise one or more of the modified nucleotides described above forsiRNA sequences. In one embodiment, the miRNA molecule comprises 2′OMenucleotides such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, or mixtures thereof.

In some embodiments, miRNA molecules may be used to silence theexpression of any of the target genes set forth above, such as, e.g.,genes associated with viral infection and survival, genes associatedwith metabolic diseases and disorders, genes associated withtumorigenesis and cell transformation, angiogenic genes, immunomodulatorgenes such as those associated with inflammatory and autoimmuneresponses, ligand receptor genes, and genes associated withneurodegenerative disorders.

In other embodiments, one or more agents that block the activity of amiRNA targeting an mRNA of interest are administered using a lipidparticle of the invention (e.g., a nucleic acid-lipid particle).Examples of blocking agents include, but are not limited to, stericblocking oligonucleotides, locked nucleic acid oligonucleotides, andMorpholino oligonucleotides. Such blocking agents may bind directly tothe miRNA or to the miRNA binding site on the target mRNA.

5. Antisense Oligonucleotides

In one embodiment, the nucleic acid is an antisense oligonucleotidedirected to a target gene or sequence of interest. The terms “antisenseoligonucleotide” or “antisense” include oligonucleotides that arecomplementary to a targeted polynucleotide sequence. Antisenseoligonucleotides are single strands of DNA or RNA that are complementaryto a chosen sequence. Antisense RNA oligonucleotides prevent thetranslation of complementary RNA strands by binding to the RNA.Antisense DNA oligonucleotides can be used to target a specific,complementary (coding or non-coding) RNA. If binding occurs, thisDNA/RNA hybrid can be degraded by the enzyme RNase H. In a particularembodiment, antisense oligonucleotides comprise from about 10 to about60 nucleotides, for example, from about 15 to about 30 nucleotides. Theterm also encompasses antisense oligonucleotides that may not be exactlycomplementary to the desired target gene. Thus, the invention can beutilized in instances where non-target specific-activities are foundwith antisense, or where an antisense sequence containing one or moremismatches with the target sequence is the most preferred for aparticular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(see, U.S. Pat. Nos. 5,739,119 and 5,759,829). Furthermore, examples ofantisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDR1), ICAM-1, E-selectin,STK-1, striatal GABAA receptor, and human EGF (see, Jaskulski et al.,Science, 240:1544-6 (1988); Vasanthakumar et al., Cancer Commun.,1:225-32 (1989); Penis et al., Brain Res Mol Brain Res., 15; 57:310-20(1998); and U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and5,610,288). Moreover, antisense constructs have also been described thatinhibit and can be used to treat a variety of abnormal cellularproliferations, e.g., cancer (see, U.S. Pat. Nos. 5,747,470; 5,591,317;and 5,783,683). The disclosures of these references are hereinincorporated by reference in their entirety for all purposes.

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Specific target regions of the mRNA include those regions at ornear the AUG translation initiation codon and those sequences that aresubstantially complementary to 5′ regions of the mRNA. These secondarystructure analyses and target site selection considerations can beperformed, for example, using v.4 of the OLIGO primer analysis software(Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithm software(Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

6. Ribozymes

According to another embodiment of the invention, nucleic acid-lipidparticles are associated with ribozymes. Ribozymes are RNA-proteincomplexes having specific catalytic domains that possess endonucleaseactivity (see, Kim et al., Proc. Natl. Acad. Sci. USA., 84:8788-92(1987); and Forster et al., Cell, 49:211-20 (1987)). For example, alarge number of ribozymes accelerate phosphoester transfer reactionswith a high degree of specificity, often cleaving only one of severalphosphoesters in an oligonucleotide substrate (see, Cech et al., Cell,27:487-96 (1981); Michel et al., J. Mol. Biol., 216:585-610 (1990);Reinhold-Hurek et al., Nature, 357:173-6 (1992)). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAmolecules are known presently. Each can catalyze the hydrolysis of RNAphosphodiester bonds in trans (and thus can cleave other RNA molecules)under physiological conditions. In general, enzymatic nucleic acids actby first binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, hepatitis 6 virus, group I intron or RNaseP RNA (in associationwith an RNA guide sequence), or Neurospora VS RNA motif, for example.Specific examples of hammerhead motifs are described in, e.g., Rossi etal., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifsare described in, e.g., EP 0360257, Hampel et al., Biochemistry,28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304 (1990);and U.S. Pat. No. 5,631,359. An example of the hepatitis 6 virus motifis described in, e.g., Perrotta et al., Biochemistry, 31:11843-52(1992). An example of the RNaseP motif is described in, e.g.,Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of theNeurospora VS RNA ribozyme motif is described in, e.g., Saville et al.,Cell, 61:685-96 (1990); Saville et al., Proc. Natl. Acad. Sci. USA,88:8826-30 (1991); Collins et al., Biochemistry, 32:2795-9 (1993). Anexample of the Group I intron is described in, e.g., U.S. Pat. No.4,987,071. Important characteristics of enzymatic nucleic acid moleculesused according to the invention are that they have a specific substratebinding site which is complementary to one or more of the target geneDNA or RNA regions, and that they have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule. Thus, the ribozyme constructs need not belimited to specific motifs mentioned herein. The disclosures of thesereferences are herein incorporated by reference in their entirety forall purposes.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in, e.g.,PCT Publication Nos. WO 93/23569 and WO 94/02595, and synthesized to betested in vitro and/or in vivo as described therein. The disclosures ofthese PCT publications are herein incorporated by reference in theirentirety for all purposes.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases(see, e.g., PCT Publication Nos. WO 92/07065, WO 93/15187, WO 91/03162,and WO 94/13688; EP 92110298.4; and U.S. Pat. No. 5,334,711, whichdescribe various chemical modifications that can be made to the sugarmoieties of enzymatic RNA molecules, the disclosures of which are eachherein incorporated by reference in their entirety for all purposes),modifications which enhance their efficacy in cells, and removal of stemII bases to shorten RNA synthesis times and reduce chemicalrequirements.

7. Immunostimulatory Oligonucleotides

Nucleic acids associated with lipid particles of the present inventionmay be immunostimulatory, including immunostimulatory oligonucleotides(ISS; single- or double-stranded) capable of inducing an immune responsewhen administered to a subject, which may be a mammal such as a human.ISS include, e.g., certain palindromes leading to hairpin secondarystructures (see, Yamamoto et al., J. Immunol., 148:4072-6 (1992)), orCpG motifs, as well as other known ISS features (such as multi-Gdomains; see; PCT Publication No. WO 96/11266, the disclosure of whichis herein incorporated by reference in its entirety for all purposes).

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target sequence in order to provoke an immuneresponse. Thus, certain immunostimulatory nucleic acids may comprise asequence corresponding to a region of a naturally-occurring gene ormRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein the CpG dinucleotide is methylated. In an alternative embodiment, thenucleic acid comprises at least two CpG dinucleotides, wherein at leastone cytosine in the CpG dinucleotides is methylated. In a furtherembodiment, each cytosine in the CpG dinucleotides present in thesequence is methylated. In another embodiment, the nucleic acidcomprises a plurality of CpG dinucleotides, wherein at least one of theCpG dinucleotides comprises a methylated cytosine. Examples ofimmunostimulatory oligonucleotides suitable for use in the compositionsand methods of the present invention are described in PCT ApplicationNo. PCT/US08/88676, filed Dec. 31, 2008, PCT Publication Nos. WO02/069369 and WO 01/15726, U.S. Pat. No. 6,406,705, and Raney et al., J.Pharm. Exper. Ther., 298:1185-92 (2001), the disclosures of which areeach herein incorporated by reference in their entirety for allpurposes. In certain embodiments, the oligonucleotides used in thecompositions and methods of the invention have a phosphodiester (“PO”)backbone or a phosphorothioate (“PS”) backbone, and/or at least onemethylated cytosine residue in a CpG motif.

8. mRNA

Certain embodiments of the invention provide compositions and methodsthat can be used to express one or more mRNA molecules in a living cell(e.g., cells within a human body). The mRNA molecules encode one or morepolypeptides that is/are expressed within the living cells. In someembodiments, the polypeptides are expressed within a diseased organism(e.g., mammal, such as a human being), and expression of the polypeptideameliorates one or more symptoms of the disease. The compositions andmethods of the invention are particularly useful for treating humandiseases caused by the absence, or reduced levels, of a functionalpolypeptide within the human body. Accordingly, an certain embodiments,an LNP may comprise one or more nucleic acid molecules, such as one ormore mRNA molecules (e.g, a cocktail of mRNA molecules).

In some embodiments, the mRNA(s) are fully encapsulated in the nucleicacid-lipid particle (e.g., LNP). With respect to formulations comprisingan mRNA cocktail, the different types of mRNA species present in thecocktail (e.g., mRNA having different sequences) may be co-encapsulatedin the same particle, or each type of mRNA species present in thecocktail may be encapsulated in a separate particle. The mRNA cocktailmay be formulated in the particles described herein using a mixture oftwo or more individual mRNAs (each having a unique sequence) atidentical, similar, or different concentrations or molar ratios. In oneembodiment, a cocktail of mRNAs (corresponding to a plurality of mRNAswith different sequences) is formulated using identical, similar, ordifferent concentrations or molar ratios of each mRNA species, and thedifferent types of mRNAs are co-encapsulated in the same particle. Inanother embodiment, each type of mRNA species present in the cocktail isencapsulated in different particles at identical, similar, or differentmRNA concentrations or molar ratios, and the particles thus formed (eachcontaining a different mRNA payload) are administered separately (e.g.,at different times in accordance with a therapeutic regimen), or arecombined and administered together as a single unit dose (e.g., with apharmaceutically acceptable carrier). The particles described herein areserum-stable, are resistant to nuclease degradation, and aresubstantially non-toxic to mammals such as humans.

a. Modifications to mRNA

mRNA used in the practice of the present invention can include one, two,or more than two nucleoside modifications. In some embodiments, themodified mRNA exhibits reduced degradation in a cell into which the mRNAis introduced, relative to a corresponding unmodified mRNA.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methy1-pseudouridine, 4-thio-1-methy 1-pseudouridine, 2-thio-1-methy1-pseudouridine, 1-methy 1-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihy drouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In specific embodiments, a modified nucleoside is5′-O-(1-Thiophosphate)-Adenosine, 5′-O-(1-Thiophosphate)-Cytidine,5′-O-(1-Thiophosphate)-Guanosine, 5′-O-(1-Thiophosphate)-Uridine or5′-O-(1-Thiophosphate)-Pseudouridine. The α-thio substituted phosphatemoiety is provided to confer stability to RNA polymers through theunnatural phosphorothioate backbone linkages. Phosphorothioate RNA haveincreased nuclease resistance and subsequently a longer half-life in acellular environment. Phosphorothioate linked nucleic acids are expectedto also reduce the innate immune response through weakerbinding/activation of cellular innate immune molecules.

In certain embodiments it is desirable to intracellularly degrade amodified nucleic acid introduced into the cell, for example if precisetiming of protein production is desired. Thus, the invention provides amodified nucleic acid containing a degradation domain, which is capableof being acted on in a directed manner within a cell.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

b. Optional Components of the Modified Nucleic Acids

In further embodiments, the modified nucleic acids may include otheroptional components, which can be beneficial in some embodiments. Theseoptional components include, but are not limited to, untranslatedregions, kozak sequences, intronic nucleotide sequences, internalribosome entry site (IRES), caps and polyA tails. For example, a 5′untranslated region (UTR) and/or a 3′ UTR may be provided, whereineither or both may independently contain one or more differentnucleoside modifications. In such embodiments, nucleoside modificationsmay also be present in the translatable region. Also provided arenucleic acids containing a Kozak sequence.

Additionally, provided are nucleic acids containing one or more intronicnucleotide sequences capable of being excised from the nucleic acid.

i. Untranslated Regions (UTRs)

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon;whereas, the 3′UTR starts immediately following the stop codon andcontinues until the transcriptional termination signal. There is growingbody of evidence about the regulatory roles played by the UTRs in termsof stability of the nucleic acid molecule and translation. Theregulatory features of a UTR can be incorporated into the mRNA used inthe present invention to increase the stability of the molecule. Thespecific features can also be incorporated to ensure controlleddown-regulation of the transcript in case they are misdirected toundesired organs sites.

ii. 5′ Capping

The 5′ cap structure of an mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

IRES Sequences

mRNA containing an internal ribosome entry site (IRES) are also usefulin the practice of the present invention. An IRES may act as the soleribosome binding site, or may serve as one of multiple ribosome bindingsites of an mRNA. An mRNA containing more than one functional ribosomebinding site may encode several peptides or polypeptides that aretranslated independently by the ribosomes (“multicistronic mRNA”). WhenmRNA are provided with an IRES, further optionally provided is a secondtranslatable region. Examples of IRES sequences that can be usedaccording to the invention include without limitation, those frompicornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV),encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),murine leukemia virus (MLV), simian immune deficiency viruses (S1V) orcricket paralysis viruses (CrPV).

iii. Poly-A Tails

During RNA processing, a long chain of adenine nucleotides (poly-A tail)may be added to a polynucleotide such as an mRNA molecules in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript may be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between 100 and 250residues long.

Generally, the length of a poly-A tail is greater than 30 nucleotides inlength. In another embodiment, the poly-A tail is greater than 35nucleotides in length (e.g., at least or greater than about 35, 40, 45,50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2,000, 2,500, and 3,000 nucleotides).

In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the modified mRNA. The poly-A tailmay also be designed as a fraction of modified nucleic acids to which itbelongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60,70, 80, or 90% or more of the total length of the modified mRNA or thetotal length of the modified mRNA minus the poly-A tail.

c. Generating mRNA Molecules

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989));as are PCR methods (see, U.S. Pat. Nos. 4,683,195 and 4,683,202; PCRProtocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Expression libraries are also well known to those of skill inthe art. Additional basic texts disclosing the general methods of use inthis invention include Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994). The disclosures of these references areherein incorporated by reference in their entirety for all purposes.

d. Encoded Polypeptides

The mRNA component of a nucleic acid-lipid particle described herein canbe used to express a polypeptide of interest. Certain diseases in humansare caused by the absence or impairment of a functional protein in acell type where the protein is normally present and active. Thefunctional protein can be completely or partially absent due, e.g., totranscriptional inactivity of the encoding gene or due to the presenceof a mutation in the encoding gene that renders the protein completelyor partially non-functional. Examples of human diseases that are causedby complete or partial inactivation of a protein include X-linked severecombined immunodeficiency (X-SCID) and adrenoleukodystrophy (X-ALD).X-SCID is caused by one or more mutations in the gene encoding thecommon gamma chain protein that is a component of the receptors forseveral interleukins that are involved in the development and maturationof B and T cells within the immune system. X-ALD is caused by one ormore mutations in a peroxisomal membrane transporter protein gene calledABCD1. Individuals afflicted with X-ALD have very high levels of longchain fatty acids in tissues throughout the body, which causes a varietyof symptoms that may lead to mental impairment or death.

Attempts have been made to use gene therapy to treat some diseasescaused by the absence or impairment of a functional protein in a celltype where the protein is normally present and active. Gene therapytypically involves introduction of a vector that includes a geneencoding a functional form of the affected protein, into a diseasedperson, and expression of the functional protein to treat the disease.Thus far, gene therapy has met with limited success. Additionally,certain aspects of delivering mRNA using LNPs have been described, e.g.,in International Publication Numbers WO 2018/006052 and WO 2015/011633.

As such, there is a continuing need for improvement for expressing afunctional form of a protein within a human who suffers from a diseasecaused by the complete or partial absence of the functional protein, andthere is a need for improved delivery of nucleic acids (e.g., mRNA) viaa methods and compositions, e.g., that can trigger less of an immuneresponse to the therapy. Certain embodiments of the present inventionare useful in this context. Thus, in certain embodiments, expression ofthe polypeptide ameliorates one or more symptoms of a disease ordisorder. Certain compositions and methods of the invention may beuseful for treating human diseases caused by the absence, or reducedlevels, of a functional polypeptide within the human body. In otherembodiments, certain compositions and methods of the invention may beuseful for expressing a vaccine antigen for treating cancer.

9. Self-Amplifying RNA

In certain embodiments, the nucleic acid is one or more self-amplifyingRNA molecules. Self-amplifying RNA (sa-RNA) may also be referred to asself-replicating RNA, replication-competent RNA, replicons or RepRNA.RepRNA, referred to as self-amplifying mRNA when derived frompositive-strand viruses, is generated from a viral genome lacking atleast one structural gene; it can translate and replicate (hence“self-amplifying”) without generating infectious progeny virus. Incertain embodiments, the RepRNA technology may be used to insert a genecassette encoding a desired antigen of interest. For example, thealphaviral genome is divided into two open reading frames (ORFs): thefirst ORF encodes proteins for the RNA dependent RNA polymerase(replicase), and the second ORF encodes structural proteins. In sa-RNAvaccine constructs, the ORF encoding viral structural proteins may bereplaced with any antigen of choice, while the viral replicase remainsan integral part of the vaccine and drives intracellular amplificationof the RNA after immunization.

B. Other Active Agents

In certain embodiments, the active agent associated with the lipidparticles of the invention may comprise one or more therapeuticproteins, polypeptides, or small organic molecules or compounds.Non-limiting examples of such therapeutically effective agents or drugsinclude oncology drugs (e.g., chemotherapy drugs, hormonal therapeuticagents, immunotherapeutic agents, radiotherapeutic agents, etc.),lipid-lowering agents, anti-viral drugs, anti-inflammatory compounds,antidepressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs such asanti-arrhythmic agents, hormones, vasoconstrictors, and steroids. Theseactive agents may be administered alone in the lipid particles of theinvention, or in combination (e.g., co-administered) with lipidparticles of the invention comprising nucleic acid, such as interferingRNA or mRNA.

Non-limiting examples of chemotherapy drugs include platinum-based drugs(e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin,satraplatin, etc.), alkylating agents (e.g., cyclophosphamide,ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine,uramustine, thiotepa, nitrosoureas, etc.), anti-metabolites (e.g.,5-fluorouracil (5-FU), azathioprine, methotrexate, leucovorin,capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine,pemetrexed, raltitrexed, etc.), plant alkaloids (e.g., vincristine,vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel(taxol), docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan(CPT-11; Camptosar), topotecan, amsacrine, etoposide (VP16), etoposidephosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin,adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), tyrosine kinase inhibitors (e.g.,gefitinib (Iressa®), sunitinib (Sutent®; SU11248), erlotinib (Tarceva®;OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib(SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib(Gleevec®; STI571), dasatinib (BMS-354825), leflunomide (SU101),vandetanib (Zactima™; ZD6474), etc.), pharmaceutically acceptable saltsthereof, stereoisomers thereof, derivatives thereof, analogs thereof,and combinations thereof.

Examples of conventional hormonal therapeutic agents include, withoutlimitation, steroids (e.g., dexamethasone), finasteride, aromataseinhibitors, tamoxifen, and goserelin as well as othergonadotropin-releasing hormone agonists (GnRH).

Examples of conventional immunotherapeutic agents include, but are notlimited to, immunostimulants (e.g., Bacillus Calmette-Guérin (BCG),levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³I,etc.).

Examples of conventional radiotherapeutic agents include, but are notlimited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi, optionally conjugated to antibodies directedagainst tumor antigens.

Additional oncology drugs that may be used according to the inventioninclude, but are not limited to, alkeran, allopurinol, altretamine,amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU,carmustine, CCNU, celecoxib, cladribine, cyclosporin A, cytosinearabinoside, cytoxan, dexrazoxane, DTIC, estramustine, exemestane,FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin,interferon, letrozole, leustatin, leuprolide, litretinoin, megastrol,L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate,Pegademase, pentostatin, porfimer sodium, prednisone, rituxan,streptozocin, STI-571, taxotere, temozolamide, VM-26, toremifene,tretinoin, ATRA, valrubicin, and velban. Other examples of oncologydrugs that may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors, and camptothecins.

Non-limiting examples of lipid-lowering agents for treating a lipiddisease or disorder associated with elevated triglycerides, cholesterol,and/or glucose include statins, fibrates, ezetimibe, thiazolidinediones,niacin, beta-blockers, nitroglycerin, calcium antagonists, fish oil, andmixtures thereof.

Examples of anti-viral drugs include, but are not limited to, abacavir,aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol,atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine,didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide,entecavir, entry inhibitors, famciclovir, fixed dose combinations,fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors,ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir,inosine, integrase inhibitors, interferon type III (e.g., IFN-λmolecules such as IFN-λ1, IFN-λ2, and IFN-λ3), interferon type II (e.g.,IFN-γ), interferon type I (e.g., IFN-α such as PEGylated IFN-α, IFN-β,IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ), interferon, lamivudine,lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir,nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir,peramivir, pleconaril, podophyllotoxin, protease inhibitors, reversetranscriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir,stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil,tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,zanamivir, zidovudine, pharmaceutically acceptable salts thereof,stereoisomers thereof, derivatives thereof, analogs thereof, andmixtures thereof.

III. LIPID PARTICLES

The lipid particles of the invention typically comprise an active agentor therapeutic agent, a cationic lipid, a non-cationic lipid, and aconjugated lipid that inhibits aggregation of particles. In someembodiments, the active agent or therapeutic agent is fully encapsulatedwithin the lipid portion of the lipid particle such that the activeagent or therapeutic agent in the lipid particle is resistant in aqueoussolution to enzymatic degradation, e.g., by a nuclease or protease. Inother embodiments, the lipid particles described herein aresubstantially non-toxic to mammals such as humans. The lipid particlesof the invention typically have a mean diameter of from about 40 nm toabout 150 nm, from about 50 nm to about 150 nm, from about 60 nm toabout 130 nm, from about 70 nm to about 110 nm, or from about 70 toabout 90 nm.

In some embodiments, the lipid particles of the invention areserum-stable nucleic acid-lipid particles (LNP) which comprise one ormore nucleic acid molecules, such as an interfering RNA (e.g., siRNA,aiRNA, and/or miRNA) or mRNA; a cationic lipid (e.g., a cationic lipidof Formulas I, II, and/or III); a non-cationic lipid (e.g., cholesterolalone or mixtures of one or more phospholipids and cholesterol); and aconjugated lipid that inhibits aggregation of the particles (e.g., oneor more PEG-lipid conjugates). The LNP may comprise at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more unmodified and/or modified nucleic acidmolecules. Nucleic acid-lipid particles and their method of preparationare described in, e.g., U.S. Pat. Nos. 5,753,613; 5,785,992; 5,705,385;5,976,567; 5,981,501; 6,110,745; and 6,320,017; and PCT Publication No.WO 96/40964, the disclosures of which are each herein incorporated byreference in their entirety for all purposes.

A. Cationic Lipids

Any of a variety of cationic lipids may be used in the lipid particlesof the invention (e.g., LNP), either alone or in combination with one ormore other cationic lipid species or non-cationic lipid species.

Cationic lipids which are useful in the present invention can be any ofa number of lipid species which carry a net positive charge atphysiological pH. Such lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3.beta.-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), and mixturesthereof. A number of these lipids and related analogs have beendescribed in U.S. Patent Publication Nos. 20060083780 and 20060240554;U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613;and 5,785,992; and PCT Publication No. WO 96/10390, the disclosures ofwhich are each herein incorporated by reference in their entirety forall purposes. Additionally, a number of commercial preparations ofcationic lipids are available and can be used in the present invention.These include, e.g., LIPOFECTIN® (commercially available cationicliposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, N.Y.,USA); LIPOFECTAMINE® (commercially available cationic liposomescomprising DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM®(commercially available cationic liposomes comprising DOGS from PromegaCorp., Madison, Wis., USA).

Additionally, cationic lipids of Formula I having the followingstructures are useful in the present invention.

wherein R¹ and R² are independently selected and are H or C₁-C₃ alkyls,R³ and R⁴ are independently selected and are alkyl groups having fromabout 10 to about 20 carbon atoms, and at least one of R³ and R⁴comprises at least two sites of unsaturation. In certain instances, R³and R⁴ are both the same, i.e., R³ and R⁴ are both linoleyl (C₁₈), etc.In certain other instances, R³ and R⁴ are different, i.e., R³ istetradectrienyl (C₁₄) and R⁴ is linoleyl (C₁₈). In one embodiment, thecationic lipid of Formula I is symmetrical, i.e., R³ and R⁴ are both thesame. In another embodiment, both R³ and R⁴ comprise at least two sitesof unsaturation. In some embodiments, R³ and R⁴ are independentlyselected from the group consisting of dodecadienyl, tetradecadienyl,hexadecadienyl, linoleyl, and icosadienyl. In one embodiment, R³ and R⁴are both linoleyl. In some embodiments, R³ and R⁴ comprise at leastthree sites of unsaturation and are independently selected from, e.g.,dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, andicosatrienyl. In particular embodiments, the cationic lipid of Formula Iis 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

Furthermore, cationic lipids of Formula II having the followingstructures are useful in the present invention.

wherein R¹ and R² are independently selected and are H or C₁-C₃ alkyls,R³ and R⁴ are independently selected and are alkyl groups having fromabout 10 to about 20 carbon atoms, and at least one of R³ and R⁴comprises at least two sites of unsaturation. In certain instances, R³and R⁴ are both the same, i.e., R³ and R⁴ are both linoleyl (C₁₈), etc.In certain other instances, R³ and R⁴ are different, i.e., R³ istetradectrienyl (C₁₄) and R⁴ is linoleyl (C₁₈). In one embodiment, thecationic lipids of the present invention are symmetrical, i.e., R³ andR⁴ are both the same. In another embodiment, both R³ and R⁴ comprise atleast two sites of unsaturation. In some embodiments, R³ and R⁴ areindependently selected from the group consisting of dodecadienyl,tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In oneembodiment, R³ and R⁴ are both linoleyl. In some embodiments, R³ and R⁴comprise at least three sites of unsaturation and are independentlyselected from, e.g., dodecatrienyl, tetradectrienyl, hexadecatrienyl,linolenyl, and icosatrienyl.

Moreover, cationic lipids of Formula III having the following structures(or salts thereof) are useful in the present invention.

wherein R¹ and R² are either the same or different and independentlyoptionally substituted C₁₂-C₂₄ alkyl, optionally substituted C₁₂-C₂₄alkenyl, optionally substituted C₁₂-C₂₄ alkynyl, or optionallysubstituted C₁₂-C₂₄ acyl; R³ and R⁴ are either the same or different andindependently optionally substituted C₁-C₆ alkyl, optionally substitutedC₁-C₆ alkenyl, or optionally substituted C₁-C₆ alkynyl or R³ and R⁴ mayjoin to form an optionally substituted heterocyclic ring of 4 to 6carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R⁵is either absent or hydrogen or C₁-C₆ alkyl to provide a quaternaryamine; m, n, and p are either the same or different and independentlyeither 0 or 1 with the proviso that m, n, and p are not simultaneously0; q is 0, 1, 2, 3, or 4; and Y and Z are either the same or differentand independently O, S, or NH.

In some embodiments, the cationic lipid of Formula III is2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2”), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane(DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane(DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane(DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane(DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane(DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane(DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), ormixtures thereof. In some embodiments, the cationic lipid of Formula IIIis DLin-K-C2-DMA (XTC2).

The cationic lipid typically comprises from about 50 mol % to about 90mol %, from about 50 mol % to about 85 mol %, from about 50 mol % toabout 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol% to about 70 mol %, from about 50 mol % to about 65 mol %, or fromabout 55 mol % to about 65 mol % of the total lipid present in theparticle.

It will be readily apparent to one of skill in the art that depending onthe intended use of the particles, the proportions of the components canbe varied and the delivery efficiency of a particular formulation can bemeasured using, e.g., an endosomal release parameter (ERP) assay.

B. Non-Cationic Lipids

The non-cationic lipids used in the lipid particles of the invention(e.g., LNP) can be any of a variety of neutral uncharged, zwitterionic,or anionic lipids capable of producing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids aretypically acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof such as cholestanol, cholestanone,cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether,cholesteryl-4′-hydroxybutyl ether, and mixtures thereof.

In some embodiments, the non-cationic lipid present in the lipidparticles (e.g., LNP) comprises or consists of cholesterol or aderivative thereof, e.g., a phospholipid-free lipid particleformulation. In other embodiments, the non-cationic lipid present in thelipid particles (e.g., LNP) comprises or consists of one or morephospholipids, e.g., a cholesterol-free lipid particle formulation. Infurther embodiments, the non-cationic lipid present in the lipidparticles (e.g., LNP) comprises or consists of a mixture of one or morephospholipids and cholesterol or a derivative thereof.

Other examples of non-cationic lipids suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

In some embodiments, the non-cationic lipid comprises from about 13 mol% to about 49.5 mol %, from about 20 mol % to about 45 mol %, from about25 mol % to about 45 mol %, from about 30 mol % to about 45 mol %, fromabout 35 mol % to about 45 mol %, from about 20 mol % to about 40 mol %,from about 25 mol % to about 40 mol %, or from about 30 mol % to about40 mol % of the total lipid present in the particle.

In certain embodiments, the cholesterol present in phospholipid-freelipid particles comprises from about 30 mol % to about 45 mol %, fromabout 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %,or from about 35 mol % to about 40 mol % of the total lipid present inthe particle. As a non-limiting example, a phospholipid-free lipidparticle may comprise cholesterol at about 37 mol % of the total lipidpresent in the particle.

In certain other embodiments, the cholesterol present in lipid particlescontaining a mixture of phospholipid and cholesterol comprises fromabout 30 mol % to about 40 mol %, from about 30 mol % to about 35 mol %,or from about 35 mol % to about 40 mol % of the total lipid present inthe particle. As a non-limiting example, a lipid particle comprising amixture of phospholipid and cholesterol may comprise cholesterol atabout 34 mol % of the total lipid present in the particle.

In further embodiments, the cholesterol present in lipid particlescontaining a mixture of phospholipid and cholesterol comprises fromabout 10 mol % to about 30 mol %, from about 15 mol % to about 25 mol %,or from about 17 mol % to about 23 mol % of the total lipid present inthe particle. As a non-limiting example, a lipid particle comprising amixture of phospholipid and cholesterol may comprise cholesterol atabout 20 mol % of the total lipid present in the particle.

In embodiments where the lipid particles contain a mixture ofphospholipid and cholesterol or a cholesterol derivative, the mixturemay comprise up to about 40, 45, 50, 55, or 60 mol % of the total lipidpresent in the particle. In certain instances, the phospholipidcomponent in the mixture may comprise from about 2 mol % to about 12 mol%, from about 4 mol % to about 10 mol %, from about 5 mol % to about 10mol %, from about 5 mol % to about 9 mol %, or from about 6 mol % toabout 8 mol % of the total lipid present in the particle. As anon-limiting example, a lipid particle comprising a mixture ofphospholipid and cholesterol may comprise a phospholipid such as DPPC orDSPC at about 7 mol % (e.g., in a mixture with about 34 mol %cholesterol) of the total lipid present in the particle. In certainother instances, the phospholipid component in the mixture may comprisefrom about 10 mol % to about 30 mol %, from about 15 mol % to about 25mol %, or from about 17 mol % to about 23 mol % of the total lipidpresent in the particle. As another non-limiting example, a lipidparticle comprising a mixture of phospholipid and cholesterol maycomprise a phospholipid such as DPPC or DSPC at about 20 mol % (e.g., ina mixture with about 20 mol % cholesterol) of the total lipid present inthe particle.

C. PEG-Conjugated Lipids of Formula (I)

The PEG-conjugated lipid of formula (I) typically comprises from about0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, fromabout 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %,from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about1.7 mol %, from about 0.9 mol % to about 1.6 mol %, from about 0.9 mol %to about 1.8 mol %, from about 1 mol % to about 1.8 mol %, from about 1mol % to about 1.7 mol %, from about 1.2 mol % to about 1.8 mol %, fromabout 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about 1.6mol %, or from about 1.4 mol % to about 1.5 mol % of the total lipidpresent in the particle.

One of ordinary skill in the art will appreciate that the concentrationof the PEG-conjugated lipids of formula (I) can be varied depending onthe lipid conjugate employed and the rate at which the nucleicacid-lipid particle is to become fusogenic.

By controlling the composition and concentration of the PEG-conjugatedlipids of formula (I), one can control the rate at which thePEG-conjugated lipid of formula (I) exchanges out of the nucleicacid-lipid particle and, in turn, the rate at which the nucleicacid-lipid particle becomes fusogenic. For instance, the rate at whichthe nucleic acid-lipid particle becomes fusogenic can be varied, forexample, by varying the concentration of the PEG-conjugated lipid offormula (I), by varying the molecular weight of the PEG, or by varyingthe chain length and degree of saturation of the acyl chain groups onthe phosphatidylethanolamine or the ceramide. In addition, othervariables including, for example, pH, temperature, ionic strength, etc.can be used to vary and/or control the rate at which the nucleicacid-lipid particle becomes fusogenic. Other methods which can be usedto control the rate at which the nucleic acid-lipid particle becomesfusogenic will become apparent to those of skill in the art upon readingthis disclosure.

IV. PREPARATION OF LIPID PARTICLES

The lipid particles of the present invention, e.g., LNP, in which anactive agent or therapeutic agent, such as an interfering RNA or mRNA,is encapsulated in a lipid bilayer and is protected from degradation,can be formed by any method known in the art including, but not limitedto, a continuous mixing method or a direct dilution process.

In some embodiments, the cationic lipids are lipids of Formula I, II,and III, or combinations thereof. In other embodiments, the non-cationiclipids are egg sphingomyelin (ESM), distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC),dipalmitoyl-phosphatidylcholine (DPPC),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,14:0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE(1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE(1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE(1,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1 trans PE(1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE(1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethyleneglycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modifieddiacylglycerols, or PEG-modified dialkyloxypropyls), cholesterol, orcombinations thereof.

In certain embodiments, the present invention provides for LNP producedvia a continuous mixing method, e.g., a process that includes providingan aqueous solution comprising a nucleic acid in a first reservoir,providing an organic lipid solution in a second reservoir, and mixingthe aqueous solution with the organic lipid solution such that theorganic lipid solution mixes with the aqueous solution so as tosubstantially instantaneously produce a liposome encapsulating thenucleic acid (e.g., interfering RNA or mRNA). This process and theapparatus for carrying this process are described in detail in U.S.Patent Publication No. 20040142025, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The action of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a liposome substantially instantaneously upon mixing. As usedherein, the phrase “continuously diluting a lipid solution with a buffersolution” (and variations) generally means that the lipid solution isdiluted sufficiently rapidly in a hydration process with sufficientforce to effectuate vesicle generation. By mixing the aqueous solutioncomprising a nucleic acid with the organic lipid solution, the organiclipid solution undergoes a continuous stepwise dilution in the presenceof the buffer solution (i.e., aqueous solution) to produce a nucleicacid-lipid particle.

The LNP formed using the continuous mixing method typically have a sizeof from about 40 nm to about 150 nm, from about 50 nm to about 150 nm,from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, orfrom about 70 nm to about 90 nm. The particles thus formed do notaggregate and are optionally sized to achieve a uniform particle size.

In another embodiment, the present invention provides for LNP producedvia a direct dilution process that includes forming a liposome solutionand immediately and directly introducing the liposome solution into acollection vessel containing a controlled amount of dilution buffer. Insome aspects, the collection vessel includes one or more elementsconfigured to stir the contents of the collection vessel to facilitatedilution. In one aspect, the amount of dilution buffer present in thecollection vessel is substantially equal to the volume of liposomesolution introduced thereto. As a non-limiting example, a liposomesolution in 45% ethanol when introduced into the collection vesselcontaining an equal volume of dilution buffer will advantageously yieldsmaller particles.

In yet another embodiment, the present invention provides for LNPproduced via a direct dilution process in which a third reservoircontaining dilution buffer is fluidly coupled to a second mixing region.In this embodiment, the liposome solution formed in a first mixingregion is immediately and directly mixed with dilution buffer in thesecond mixing region. In some aspects, the second mixing region includesa T-connector arranged so that the liposome solution and the dilutionbuffer flows meet as opposing 1800 flows; however, connectors providingshallower angles can be used, e.g., from about 270 to about 180°. A pumpmechanism delivers a controllable flow of buffer to the second mixingregion. In one aspect, the flow rate of dilution buffer provided to thesecond mixing region is controlled to be substantially equal to the flowrate of liposome solution introduced thereto from the first mixingregion. This embodiment advantageously allows for more control of theflow of dilution buffer mixing with the liposome solution in the secondmixing region, and therefore also the concentration of liposome solutionin buffer throughout the second mixing process. Such control of thedilution buffer flow rate advantageously allows for small particle sizeformation at reduced concentrations.

These processes and the apparatuses for carrying out these directdilution processes are described in detail in U.S. Patent PublicationNo. 20070042031, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

The LNP formed using the direct dilution process typically have a sizeof from about 40 nm to about 150 nm, from about 50 nm to about 150 nm,from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, orfrom about 70 nm to about 90 nm. The particles thus formed do notaggregate and are optionally sized to achieve a uniform particle size.

If needed, the lipid particles of the invention (e.g., LNP) can be sizedby any of the methods available for sizing liposomes. The sizing may beconducted in order to achieve a desired size range and relatively narrowdistribution of particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles, is described in U.S. Pat. No. 4,737,323, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. Sonicating a particle suspension either by bath orprobe sonication produces a progressive size reduction down to particlesof less than about 50 nm in size. Homogenization is another method whichrelies on shearing energy to fragment larger particles into smallerones. In a typical homogenization procedure, particles are recirculatedthrough a standard emulsion homogenizer until selected particle sizes,typically between about 60 and about 80 nm, are observed. In bothmethods, the particle size distribution can be monitored by conventionallaser-beam particle size discrimination, or QELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In some embodiments, the nucleic acids in the LNP are precondensed asdescribed in, e.g., U.S. patent application Ser. No. 09/744,103, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

In other embodiments, the methods will further comprise adding non-lipidpolycations which are useful to effect the lipofection of cells usingthe present compositions. Examples of suitable non-lipid polycationsinclude, hexadimethrine bromide (sold under the brandname POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine, and polyethyleneimine. Addition of these salts istypically after the particles have been formed.

In some embodiments, the nucleic acid to lipid ratios (mass/mass ratios)in a formed LNP will range from about 0.01 to about 0.2, from about 0.02to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about0.08. The ratio of the starting materials also falls within this range.In other embodiments, the LNP preparation uses about 400 μg nucleic acidper 10 mg total lipid or a nucleic acid to lipid mass ratio of about0.01 to about 0.08 and, for example, about 0.04, which corresponds to1.25 mg of total lipid per 50 μg of nucleic acid. In other embodiments,the particle has a nucleic acid:lipid mass ratio of about 0.08.

In other embodiments, the lipid to nucleic acid ratios (mass/massratios) in a formed LNP will range from about 1 (1:1) to about 100(100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1) toabout 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3(3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), fromabout 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1),from about 2 (2:1) to about 25 (25:1), from about 3 (3:1) to about 25(25:1), from about 4 (4:1) to about 25 (25:1), from about 5 (5:1) toabout 50 (50:1), from about 5 (5:1) to about 25 (25:1), from about 5(5:1) to about 20 (20:1), from about 5 (5:1) to about 15 (15:1), fromabout 5 (5:1) to about 10 (10:1), about 5 (5:1), 6 (6:1), 7 (7:1), 8(8:1), 9 (9:1), (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), 15(15:1), 16 (16:1), 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21(21:1), 22 (22:1), 23 (23:1), 24 (24:1), 25 (25:1), 26 (26:1), 27(27:1), 28 (28:1), 29 (29:1) or 30 (30:1). The ratio of the startingmaterials also falls within this range.

V. KITS

The present invention also provides lipid particles (e.g., LNP) in kitform. The kit may comprise a container which is compartmentalized forholding the various elements of the lipid particles (e.g., the activeagents or therapeutic agents such as nucleic acids and the individuallipid components of the particles). In some embodiments, the kit mayfurther comprise an endosomal membrane destabilizer (e.g., calciumions). The kit typically contains the lipid particle compositions of thepresent invention, typically in dehydrated form, with instructions fortheir rehydration and administration.

As explained herein, the lipid particles of the invention (e.g., LNP)can be tailored to target particular tissues, organs, or tumors ofinterest. In certain instances, targeting of lipid particles such as LNPmay be carried out by controlling the composition of the particleitself. For instance, as set forth in Example 11, it has been found thatthe 1:57 PEG-cDSA LNP formulation can be used to target tumors outsideof the liver, whereas the 1:57 PEG-cDMA LNP formulation can be used totarget the liver (including liver tumors).

In certain other instances, it may be desirable to have a targetingmoiety attached to the surface of the lipid particle to further enhancethe targeting of the particle. Methods of attaching targeting moieties(e.g., antibodies, proteins, etc.) to lipids (such as those used in thepresent particles) are known to those of skill in the art.

VI. ADMINISTRATION OF LIPID PARTICLES

Once formed, the lipid particles of the invention (e.g., LNP) are usefulfor the introduction of active agents or therapeutic agents (e.g.,nucleic acids, such as interfering RNA or mRNA) into cells. Accordingly,the present invention also provides methods for introducing an activeagent or therapeutic agent such as a nucleic acid (e.g., interfering RNAor mRNA) into a cell. The methods are carried out in vitro or in vivo byfirst forming the particles as described above and then contacting theparticles with the cells for a period of time sufficient for delivery ofthe active agent or therapeutic agent to the cells to occur.

The lipid particles of the invention (e.g., LNP) can be adsorbed toalmost any cell type with which they are mixed or contacted. Onceadsorbed, the particles can either be endocytosed by a portion of thecells, exchange lipids with cell membranes, or fuse with the cells.Transfer or incorporation of the active agent or therapeutic agent(e.g., nucleic acid) portion of the particle can take place via any oneof these pathways. In particular, when fusion takes place, the particlemembrane is integrated into the cell membrane and the contents of theparticle combine with the intracellular fluid.

The lipid particles of the invention (e.g., LNP) can be administeredeither alone or in a mixture with a pharmaceutically-acceptable carrier(e.g., physiological saline or phosphate buffer) selected in accordancewith the route of administration and standard pharmaceutical practice.Generally, normal buffered saline (e.g., 135-150 mM NaCl) will beemployed as the pharmaceutically-acceptable carrier. Other suitablecarriers include, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like, including glycoproteins for enhanced stability,such as albumin, lipoprotein, globulin, etc. Additional suitablecarriers are described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES,Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As usedherein, “carrier” includes any and all solvents, dispersion media,vehicles, coatings, diluents, antibacterial and antifungal agents,isotonic and absorption delaying agents, buffers, carrier solutions,suspensions, colloids, and the like. The phrase“pharmaceutically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human.

The pharmaceutically-acceptable carrier is generally added followingparticle formation. Thus, after the particle is formed, the particle canbe diluted into pharmaceutically-acceptable carriers such as normalbuffered saline.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2 to 5%, to as much as about 10 to 90% by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration.

The pharmaceutical compositions of the present invention may besterilized by conventional, well-known sterilization techniques. Aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically-acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, andcalcium chloride. Additionally, the particle suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

A. In Vivo Administration

Systemic delivery for in vivo therapy, e.g., delivery of a therapeuticnucleic acid to a distal target cell via body systems such as thecirculation, has been achieved using nucleic acid-lipid particles suchas those described in PCT Publication Nos. WO 05/007196, WO 05/121348,WO 05/120152, and WO 04/002453, the disclosures of which are hereinincorporated by reference in their entirety for all purposes. Thepresent invention also provides fully encapsulated lipid particles thatprotect the nucleic acid from nuclease degradation in serum, arenonimmunogenic, are small in size, and are suitable for repeat dosing.

For in vivo administration, administration can be in any manner known inthe art, e.g., by injection, oral administration, inhalation (e.g.,intransal or intratracheal), transdermal application, or rectaladministration. Administration can be accomplished via single or divideddoses. The pharmaceutical compositions can be administered parenterally,i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In some embodiments, thepharmaceutical compositions are administered intravenously orintraperitoneally by a bolus injection (see, e.g., U.S. Pat. No.5,286,634). Intracellular nucleic acid delivery has also been discussedin Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino etal., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. DrugCarrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).Still other methods of administering lipid-based therapeutics aredescribed in, for example, U.S. Pat. Nos. 3,993,754; 4,145,410;4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles canbe administered by direct injection at the site of disease or byinjection at a site distal from the site of disease (see, e.g., Culver,HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71 (1994)). The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering nucleic acid compositions directly tothe lungs via nasal aerosol sprays have been described, e.g., in U.S.Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs usingintranasal microparticle resins and lysophosphatidyl-glycerol compounds(U.S. Pat. No. 5,725,871) are also well-known in the pharmaceuticalarts. Similarly, transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045. The disclosures of the above-described patents are hereinincorporated by reference in their entirety for all purposes.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions are typicallyadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically, or intrathecally.

Generally, when administered intravenously, the lipid particleformulations are formulated with a suitable pharmaceutical carrier. Manypharmaceutically acceptable carriers may be employed in the compositionsand methods of the present invention. Suitable formulations for use inthe present invention are found, for example, in REMINGTON'SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). A variety of aqueous carriers may be used, for example,water, buffered water, 0.4% saline, 0.3% glycine, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Generally, normal buffered saline (135-150mM NaCl) will be employed as the pharmaceutically acceptable carrier,but other suitable carriers will suffice. These compositions can besterilized by conventional liposomal sterilization techniques, such asfiltration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. Thesecompositions can be sterilized using the techniques referred to aboveor, alternatively, they can be produced under sterile conditions. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles disclosed herein may bedelivered via oral administration to the individual. The particles maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, pills, lozenges, elixirs,mouthwash, suspensions, oral sprays, syrups, wafers, and the like (see,e.g., U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes). These oral dosage forms may also contain thefollowing: binders, gelatin; excipients, lubricants, and/or flavoringagents. When the unit dosage form is a capsule, it may contain, inaddition to the materials described above, a liquid carrier. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. Of course, any material used inpreparing any unit dosage form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed.

Typically, these oral formulations may contain at least about 0.1% ofthe lipid particles or more, although the percentage of the particlesmay, of course, be varied and may conveniently be between about 1% or 2%and about 60% or 70% or more of the weight or volume of the totalformulation. Naturally, the amount of particles in each therapeuticallyuseful composition may be prepared is such a way that a suitable dosagewill be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

Formulations suitable for oral administration can consist of: (a) liquidsolutions, such as an effective amount of a packaged therapeutic agentsuch as nucleic acid (e.g., interfering RNA or mRNA) suspended indiluents such as water, saline, or PEG 400; (b) capsules, sachets, ortablets, each containing a predetermined amount of a therapeutic agentsuch as nucleic acid (e.g., interfering RNA or mRNA), as liquids,solids, granules, or gelatin; (c) suspensions in an appropriate liquid;and (d) suitable emulsions. Tablet forms can include one or more oflactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch,potato starch, microcrystalline cellulose, gelatin, colloidal silicondioxide, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise atherapeutic agent such as nucleic acid (e.g., interfering RNA or mRNA)in a flavor, e.g., sucrose, as well as pastilles comprising thetherapeutic agent in an inert base, such as gelatin and glycerin orsucrose and acacia emulsions, gels, and the like containing, in additionto the therapeutic agent, carriers known in the art.

In another example of their use, lipid particles can be incorporatedinto a broad range of topical dosage forms. For instance, a suspensioncontaining nucleic acid-lipid particles such as LNP can be formulatedand administered as gels, oils, emulsions, topical creams, pastes,ointments, lotions, foams, mousses, and the like.

When preparing pharmaceutical preparations of the lipid particles of theinvention, it may be beneficial to use quantities of the particles whichhave been purified to reduce or eliminate empty particles or particleswith therapeutic agents such as nucleic acid associated with theexternal surface.

The methods of the present invention may be practiced in a variety ofhosts. Specific hosts include mammalian species, such as primates (e.g.,humans and chimpanzees as well as other nonhuman primates), canines,felines, equines, bovines, ovines, caprines, rodents (e.g., rats andmice), lagomorphs, and swine.

The amount of particles administered will depend upon the ratio oftherapeutic agent (e.g., nucleic acid) to lipid, the particulartherapeutic agent (e.g., nucleic acid) used, the disease or disorderbeing treated, the age, weight, and condition of the patient, and thejudgment of the clinician, but will generally be between about 0.01 andabout 50 mg per kilogram of body weight, typically between about 0.1 andabout 5 mg/kg of body weight, or about 108-10¹⁰ particles peradministration (e.g., injection).

B. In Vitro Administration

For in vitro applications, the delivery of therapeutic agents such asnucleic acids (e.g., interfering RNA or mRNA) can be to any cell grownin culture, whether of plant or animal origin, vertebrate orinvertebrate, and of any tissue or type. In one embodiment, the cellsare animal cells, for example, mammalian cells, such as human cells.

Contact between the cells and the lipid particles, when carried out invitro, takes place in a biologically compatible medium. Theconcentration of particles varies widely depending on the particularapplication, but is generally between about 1 μmol and about 10 mmol.Treatment of the cells with the lipid particles is generally carried outat physiological temperatures (about 37° C.) for periods of time of fromabout 1 to 48 hours, typically of from about 2 to 4 hours.

In one group of embodiments, a lipid particle suspension is added to60-80% confluent plated cells having a cell density of from about 10³ toabout 10⁵ cells/ml, more typically about 2×10⁴ cells/ml. Theconcentration of the suspension added to the cells is typically of fromabout 0.01 to 0.2 μg/ml, for example, about 0.1 μg/ml.

Using an Endosomal Release Parameter (ERP) assay, the deliveryefficiency of the LNP or other lipid particle of the invention can beoptimized. An ERP assay is described in detail in U.S. PatentPublication No. 20030077829, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Moreparticularly, the purpose of an ERP assay is to distinguish the effectof various cationic lipids and helper lipid components of LNP based ontheir relative effect on binding/uptake or fusion with/destabilizationof the endosomal membrane. This assay allows one to determinequantitatively how each component of the LNP or other lipid particleaffects delivery efficiency, thereby optimizing the LNP or other lipidparticle. Usually, an ERP assay measures expression of a reporterprotein (e.g., luciferase, (3-galactosidase, green fluorescent protein(GFP), etc.), and in some instances, a LNP formulation optimized for anexpression plasmid will also be appropriate for encapsulating aninterfering RNA or mRNA. In other instances, an ERP assay can be adaptedto measure downregulation of transcription or translation of a targetsequence in the presence or absence of an interfering RNA (e.g., siRNA).In other instances, an ERP assay can be adapted to measure theexpression of a target protein in the presence or absence of an mRNA. Bycomparing the ERPs for each of the various LNP or other lipid particles,one can readily determine the optimized system, e.g., the LNP or otherlipid particle that has the greatest uptake in the cell.

C. Cells for Delivery of Lipid Particles

The compositions and methods of the present invention are used to treata wide variety of cell types, in vivo and in vitro. Suitable cellsinclude, e.g., hematopoietic precursor (stem) cells, fibroblasts,keratinocytes, hepatocytes, endothelial cells, skeletal and smoothmuscle cells, osteoblasts, neurons, quiescent lymphocytes, terminallydifferentiated cells, slow or noncycling primary cells, parenchymalcells, lymphoid cells, epithelial cells, bone cells, and the like. Inone embodiment, an active agent or therapeutic agent, such as one ormore nucleic acid molecules (e.g, an interfering RNA (e.g., siRNA) ormRNA) is delivered to cancer cells such as, e.g., lung cancer cells,colon cancer cells, rectal cancer cells, anal cancer cells, bile ductcancer cells, small intestine cancer cells, stomach (gastric) cancercells, esophageal cancer cells, gallbladder cancer cells, liver cancercells, pancreatic cancer cells, appendix cancer cells, breast cancercells, ovarian cancer cells, cervical cancer cells, prostate cancercells, renal cancer cells, cancer cells of the central nervous system,glioblastoma tumor cells, skin cancer cells, lymphoma cells,choriocarcinoma tumor cells, head and neck cancer cells, osteogenicsarcoma tumor cells, and blood cancer cells.

In vivo delivery of lipid particles such as LNP encapsulating one ormore nucleic acid molecules (e.g., interfering RNA (e.g., siRNA) ormRNA) is suited for targeting cells of any cell type. The methods andcompositions can be employed with cells of a wide variety ofvertebrates, including mammals, such as, e.g., canines, felines,equines, bovines, ovines, caprines, rodents (e.g., mice, rats, andguinea pigs), lagomorphs, swine, and primates (e.g. monkeys,chimpanzees, and humans).

To the extent that tissue culture of cells may be required, it iswell-known in the art. For example, Freshney, Culture of Animal Cells, aManual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchleret al., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977), and the references cited thereinprovide a general guide to the culture of cells. Cultured cell systemsoften will be in the form of monolayers of cells, although cellsuspensions are also used.

D. Detection of Lipid Particles

In some embodiments, the lipid particles of the present invention (e.g.,LNP) are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7, 8 ormore hours. In other embodiments, the lipid particles of the presentinvention (e.g., LNP) are detectable in the subject at about 8, 12, 24,48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24,25, or 28 days after administration of the particles. The presence ofthe particles can be detected in the cells, tissues, or other biologicalsamples from the subject. The particles may be detected, e.g., by directdetection of the particles, detection of a therapeutic nucleic acid,such as an interfering RNA (e.g., siRNA) sequence or mRNA sequence,detection of a target sequence of interest (i.e., by detecting changesin expression of the sequence of interest), or a combination thereof.

1. Detection of Particles

Lipid particles of the invention such as LNP can be detected using anymethod known in the art. For example, a label can be coupled directly orindirectly to a component of the lipid particle using methods well-knownin the art. A wide variety of labels can be used, with the choice oflabel depending on sensitivity required, ease of conjugation with thelipid particle component, stability requirements, and availableinstrumentation and disposal provisions. Suitable labels include, butare not limited to, spectral labels such as fluorescent dyes (e.g.,fluorescein and derivatives, such as fluorescein isothiocyanate (FITC)and Oregon Green™; rhodamine and derivatives such Texas red,tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin,phycoerythrin, AMCA, CyDyes™, and the like; radiolabels such as ³H,¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.; enzymes such as horse radish peroxidase,alkaline phosphatase, etc.; spectral colorimetric labels such ascolloidal gold or colored glass or plastic beads such as polystyrene,polypropylene, latex, etc. The label can be detected using any meansknown in the art.

2. Detection of Nucleic Acids

Nucleic acids (e.g., interfering RNA or mRNA) are detected andquantified herein by any of a number of means well-known to those ofskill in the art. The detection of nucleic acids may proceed bywell-known methods such as Southern analysis, Northern analysis, gelelectrophoresis, PCR, radiolabeling, scintillation counting, andaffinity chromatography. Additional analytic biochemical methods such asspectrophotometry, radiography, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), and hyperdiffusion chromatography may alsobe employed.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in, e.g., “Nucleic Acid Hybridization, A PracticalApproach,” Eds. Hames and Higgins, IRL Press (1985).

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification methods, including the polymerase chain reaction (PCR) theligase chain reaction (LCR), Qβ-replicase amplification and other RNApolymerase mediated techniques (e.g., NASBA™) are found in Sambrook etal., In Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2000); and Ausubel et al., SHORT PROTOCOLS INMOLECULAR BIOLOGY, eds., Current Protocols, Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc. (2002); as well as U.S.Pat. No. 4,683,202; PCR Protocols, A Guide to Methods and Applications(Innis et al. eds.) Academic Press Inc. San Diego, Calif. (1990);Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research,3:81 (1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989);Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell etal., J. Clin. Chem., 35:1826 (1989); Landegren et al., Science, 241:1077(1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene,4:560 (1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan andMalek, Biotechnology, 13:563 (1995). Improved methods of cloning invitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039.Other methods described in the art are the nucleic acid sequence basedamplification (NASBA™, Cangene, Mississauga, Ontario) and Qβ-replicasesystems. These systems can be used to directly identify mutants wherethe PCR or LCR primers are designed to be extended or ligated only whena select sequence is present. Alternatively, the select sequences can begenerally amplified using, for example, nonspecific PCR primers and theamplified target region later probed for a specific sequence indicativeof a mutation. The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

Nucleic acids for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as inhibitor components aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage et al.,Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an automatedsynthesizer, as described in Needham VanDevanter et al., Nucleic AcidsRes., 12:6159 (1984). Purification of polynucleotides, where necessary,is typically performed by either native acrylamide gel electrophoresisor by anion exchange HPLC as described in Pearson et al., J. Chrom.,255:137 149 (1983). The sequence of the synthetic polynucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology, 65:499.

An alternative means for determining the level of transcription is insitu hybridization. In situ hybridization assays are well-known and aregenerally described in Angerer et al., Methods Enzymol., 152:649 (1987).In an in situ hybridization assay, cells are fixed to a solid support,typically a glass slide. If DNA is to be probed, the cells are denaturedwith heat or alkali. The cells are then contacted with a hybridizationsolution at a moderate temperature to permit annealing of specificprobes that are labeled. The probes are typically labeled withradioisotopes or fluorescent reporters.

VII. EXAMPLES

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1

a. Preparation of Compound 1

To a solution of polyethylene glycol 2000 (14.0 g, 7.0 mmol) inanhydrous acetonitrile (100 mL) was added triethylamine (2.5 mL, 8.8mmol) and DSC (2.3 g, 8.8 mmol). The solution was stirred overnight atroom temperature then concentrated to dryness. The residue was dissolvedin dichloromethane (400 mL) and washed with saturated sodium bicarbonate(3×150 mL). The dichloromethane layer was dried on magnesium sulfate andfiltered. To this solution was added2,3-bis(tetradecyloxy)propan-1-amine (4.2 g, 7.0 mmol, U.S. Pat. No.7,803,397 B2) and triethylamine (1.5 mL, 8.8 mmol). Upon completion, thereaction mixture was concentrated to dryness and then purified by columnchromatography using a gradient of a stock solution from 30 to 70% indichloromethane to isolate the desymmetrized PEG (Stock solution: 7%MeOH, 1% ammonium hydroxide in dichloromethane). R_(f) 0.75 (TLC, 15%MeOH in DCM)

b. Preparation of Compound 2

To a solution ofpolyethyleneglycol₂₀₀₀-(2,3-bis(tetradecyloxy)propyl)carbamate (1.8 g,0.72 mmol) and triethylamine (0.4 mL, 2.9 mmol) in acetonitrile (25 mL)and dichloromethane (25 mL) was added DSC (370 mg, 1.4 mmol). Thesolution was stirred overnight at room temperature. The reaction was notcomplete by TLC (15% methanol in DCM) so additional DSC (370 mg, 1.4mmol) and TEA (0.4 mL, 2.9 mmol) was added. The solution was stirred for2 hours at room temperature then diluted with dichloromethane (100 mL),washed with saturated sodium bicarbonate (100 mL), dried on magnesiumsulfate, filtered and concentrated to dryness to afford a colorlesssolid. The product was used in the next step without furtherpurification. R_(f) 0.8 (TLC, 15% MeOH in DCM)

c. Preparation of Compound 3

To a solution of compound 2 (7.0 g, 2.5 mmol), methyl 6-aminohexanoatehydrochloride (0.50 g, 2.7 mmol) and triethylamine (0.91 g, 9.0 mmol) indichloromethane (50 mL) was refluxed for 2 hours then another aliquot ofmethyl 6-aminohexanoate hydrochloride (1.0 g, 5.4 mmol) andtriethylamine (2.5 mL) were added and reflux was continued overnight.Upon completion, the solution was concentrated to dryness and theresidue was purified by column chromatography (Gradient: 100% DCM to 5%MeOH in DCM) to afford compound 3 as a colorless solid (6.6 g, 89%).R_(f) 0.45 (TLC, 10% MeOH in DCM).

d. Preparation of Compound 4

To a solution of compound 3 (6.6 g, 2.4 mmol) in dioxane (45 mL) wasadded lithium hydroxide (1.3 g, 55.4 mmol) and water (40 mL). Thesolution was stirred overnight at room temperature then concentrated toremove the dioxane. The remaining aqueous solution was made acidic with1M HCl then extracted with dichloromethane (3×100 mL). The combinedextracts were dried on magnesium sulfate, filtered and concentrated todryness. The residue was purified by column chromatography (Gradient 2.5to 20% MeOH in DCM) to afforded compound 4 as a colorless solid (3.5 g,53%). R_(f) 0.35 (TLC, 10% MeOH in DCM). ¹H NMR (400 MHz, Chloroform-d)δ 5.15 (s, 1H), 4.28-4.13 (m, 3H), 3.82 (dd, J=5.4, 4.5 Hz, 1H),3.75-3.53 (m, 163H), 3.54-3.34 (m, 8H), 3.29-3.10 (m, 3H), 2.25 (br s,3H), 1.26 (s, 52H), 0.96-0.78 (m, 6H).

Example 2

a. Preparation of Compound 6

A solution of compound 2 (1.9 g, 0.72 mmol), compound 5 (716 mg, 1.7mmol) and triethylamine (0.3 mL, 2.2 mmol) in dichloromethane (75 mL)was stirred for 72 h at room temperature. Upon completion, the reactionmixture was diluted with dichloromethane (75 mL) washed with saturatedsodium bicarbonate. The solution was dried on magnesium sulfate,filtered and concentrated to dryness. The residue was purified by columnchromatography on silica gel (5% MeOH in DCM) to afford compound 6 as acolorless solid. R_(f) 0.7 (TLC, 15% MeOH in DCM).

b. Preparation of Compound 7

A solution of compound 6 (1.0 g) in formic acid (25 mL) was stirredovernight at room temperature. Upon completion the solution wasconcentrated to dryness and dissolved in water (40 mL) and then themicelles were subjected to tangential flow ultrafiltration (TFU, 100,000MWCO) and exchanged with water (500 mL). The solution was concentratedon the TFU to ˜10 mL then lyophilized to afford a colorless solid. R_(f)0.3 (TLC, 15% MeOH in DCM). 1H NMR (400 MHz, Chloroform-d) δ 5.14 (s,1H), 5.00 (s, 1H), 4.28-4.10 (m, 4H), 3.85-3.77 (m, 1H), 3.70-3.50 (m,158H), 3.60-3.50 (m, 2H), 3.51-3.36 (m, 8H), 3.26-3.15 (m, 1H), 2.32 (t,J=7.6 Hz, 5H), 1.99 (t, J=7.5 Hz, 5H), 1.60-1.46 (m, 4H), 1.35-1.18 (m,41H), 0.91-0.83 (m, 6H).

Example 3

Lipid stocks were prepared (about 7 mg/mL total lipid content) in 100%ethanol, using the lipid identities and molar ratios described. The mRNAwas diluted in acetate pH 5 and nuclease-free water to reach aconcentration of 0.366 mg/mL mRNA in 100 mM acetate pH 5. Equal volumesof each solution were blended at 400 mL/min in a T-connector, anddiluted with about 4 volumes of PBS, pH 7.4. Formulations were thenplaced in Slide-A-Lyzer dialysis units (MWCO 10,000) and were dialyzedovernight 10 mM Tris, 500 mM NaCl pH 8 (Tris/NaCl buffer). Followingdialysis the formulations were concentrated to about 0.6 mg/mL usingVivaSpin concentrator units (MWCO 100,000) and then filtered through a0.2 um syringe filter.

Example 3a

A composition was generated comprising the following components:

(a) nucleic acid;(b) a mixture of cholesterol and DSPC;(c) a conjugated lipid of formula:

wherein n is selected so that the resulting polymer chain has amolecular weight of about 2000 daltons; and(d) a cationic lipid of formula:

wherein the conjugated lipid comprised about 1.5 mol % of the totallipid present in the particle; DSPC comprised about 10.0 mol % of thetotal lipid present in the particle; cholesterol comprised about 38.5mol % of the total lipid present in the particle; and the cationic lipidcomprised about 50.0 mol % of the total lipid present in the particle;and

wherein the lipid to nucleic acid ratio was about 19.6.

Example 3b

A composition was generated comprising the following components:

(a) nucleic acid;(b) a mixture of cholesterol and DSPC;(c) a conjugated lipid of formula:

wherein n is selected so that the resulting polymer chain has amolecular weight of about 2000 daltons; and(d) a cationic lipid of formula:

wherein the conjugated lipid comprised about 2.0 mol % of the totallipid present in the particle; DSPC comprised about 10.0 mol % of thetotal lipid present in the particle; cholesterol comprised about 48.0mol % of the total lipid present in the particle; and the cationic lipidcomprised about 40.0 mol % of the total lipid present in the particle;and wherein the lipid to nucleic acid ratio was about 19.3.

Example 3c

A composition was generated comprising the following components:

(a) nucleic acid;(b) a mixture of cholesterol and DSPC;(c) a conjugated lipid of formula:

wherein n is selected so that the resulting polymer chain has amolecular weight of about 2000 daltons; and(d) a cationic lipid of formula:

wherein the conjugated lipid comprised about 1.6 mol % of the totallipid present in the particle; DSPC comprised about 10.9 mol % of thetotal lipid present in the particle; cholesterol comprised about 32.8mol % of the total lipid present in the particle; and the cationic lipidcomprised about 54.9 mol % of the total lipid present in the particle;and wherein the lipid to nucleic acid ratio was about 20.2.

Example 4

Generally, the LNP were injected intravenously at 0.5 mg/kg to femaleBalb/C mice, 5-8 weeks old and blood was collected at 4-6 hours postdosing; blood is collected into K2EDTA and processed to plasma, thenstored frozen at −80° C. until analysis. Activity was assayed by testingthe plasma for human EPO expression using an human EPO ELISA kit eitherfrom StemCell (catalogue #01630) or R&D Systems (catalogue DEP00)following the manufacturer's instructions. Data is provided in thefollowing Table.

As can be seen from the data in Table 1, the composition of the presentinvention is considerably more potent than the MC3 composition used inpatisiran (Onpattro), an approved LNP product for treatment of TTRamyloidosis.

TABLE 1 Efficacy of 0.5 mg/kg LNP of the present invention in aformulation containing human EPO mRNA 4 h Following IV Dosing in Balb/CMice (n = 4) Treatment EPO (mU/mL) Stdev (mU/mL) Patisiran Compositionwith 42230 2697 MC3 (1.5:50.0:38.5:10.0) Exemplary 3a Composition 11673121168

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reading the above description. The scopeof the invention should, therefore, be determined not with reference tothe above description, but should instead be determined with referenceto the appended claims, along with the full scope of equivalents towhich such claims are entitled. The disclosures of all articles andreferences, including patent applications, patents, PCT publications,and Genbank Accession Nos., are incorporated herein by reference for allpurposes.

What is claimed is:
 1. A compound of formula (I):A-B-C  (I) wherein: A is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkylthio, or (C₂-C₆)alkanoyloxy, wherein any (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkylthio, and (C₂-C₆)alkanoyloxy is substituted with one or moreanionic precursor groups, and wherein any (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkylthio, and (C₂-C₆)alkanoyloxy is optionally substituted withone or more groups independently selected from the group consisting ofhalo, hydroxyl, (C₁-C₃)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₃)alkoxycarbonyl,(C₁-C₃)alkylthio, or (C₂-C₃)alkanoyloxy; B is a polyethylene glycolchain having a molecular weight of from about 550 daltons to about10,000 daltons; C is -L-R^(a) L is selected from the group consisting ofa direct bond, —C(O)O—, —C(O)NR^(b)—, —NR^(b)—, —C(O)—, —NR^(b)C(O)O—,—NR^(b)C(O)NR^(b)—, —S—S—, —O—, —(O)CCH₂CH₂C(O)—, and—NHC(O)CH₂CH₂C(O)NH—; R^(a) is a branched (C₁₀-C₅₀)alkyl or branched(C₁₀-C₅₀)alkenyl wherein one or more carbon atoms of the branched(C₁₀-C₅₀)alkyl or branched (C₁₀-C₅₀)alkenyl have been replaced with —O—;and each R^(b) is independently H or (C₁-C₆)alkyl.
 2. The compound ofclaim 1, wherein A is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl,or (C₂-C₆)alkynyl, wherein any (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,(C₂-C₆)alkenyl, and (C₂-C₆)alkynyl is substituted with one or moreanionic precursor groups, and wherein any (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl, and (C₂-C₆)alkynyl is optionallysubstituted with one or more groups independently selected from thegroup consisting of halo, hydroxyl, (C₁-C₃)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₃)alkoxycarbonyl, (C₁-C₃)alkylthio, or (C₂-C₃)alkanoyloxy.
 3. Thecompound of claim 1, wherein A is (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,(C₂-C₆)alkenyl, or (C₂-C₆)alkynyl, wherein any (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₂-C₆)alkenyl, and (C₂-C₆)alkynyl is substitutedwith one or more anionic precursor groups.
 4. The compound of claim 1,wherein A is (C₁-C₆)alkyl that is substituted with one or more anionicprecursor groups, and is optionally substituted with one or more groupsindependently selected from the group consisting of halo, hydroxyl,(C₁-C₃)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₃)alkoxycarbonyl, (C₁-C₃)alkylthio,or (C₂-C₃)alkanoyloxy.
 5. The compound of claim 1, wherein A is(C₁-C₆)alkyl that is substituted with one or more anionic precursorgroups.
 6. The compound of any one of claims 1-5, wherein A issubstituted with one anionic precursor group.
 7. The compound of any oneof claims 1-5, wherein A is substituted with two anionic precursorgroups.
 8. The compound of any one of claims 1-5, wherein A issubstituted with three anionic precursor groups.
 9. The compound of anyone of claims 1-8, wherein each anionic precursor group is selected fromthe group consisting of —CO₂H, —O—P(═O)(OH)₂, —OS(═O)₂(OH),—O—S(═O)(OH), and —B(OH)₂.
 10. The compound of any one of claims 1-8,wherein each anionic precursor group is —CO₂H.
 11. The compound of anyone of claims 1-10, wherein B is a polyethylene glycol chain having anaverage molecular weight ranging of about 500 daltons to about 5,000daltons.
 12. The compound of any one of claims 1-10, wherein B is apolyethylene glycol chain having an average molecular weight ranging ofabout 750 daltons to about 3,000 daltons.
 13. The compound of any one ofclaims 1-10, wherein B is a polyethylene glycol chain having an averagemolecular weight range of about 2,000 daltons.
 14. The compound of anyone of claims 1-13, wherein B is linked to C directly.
 15. The compoundof any one of claims 1-13, wherein B is linked to C directly via alinker moiety.
 16. The compound of any one of claims 1-13, wherein B islinked to C through a group selected from the group consisting of—C(O)NH—, —NR—, —C(O)—, —NHC(O)O—, —NHC(O)NH—, —S—S—, —O—,—(O)CCH₂CH₂C(O)—, and —NHC(O)CH₂CH₂C(O)NH—, wherein R is H or(C₁-C₆)alkyl.
 17. The compound of any one of claims 1-16, wherein A islinked to B directly.
 18. The compound of any one of claims 1-16,wherein A is linked to B directly via a linker moiety.
 19. The compoundof any one of claims 1-16, wherein A is linked to B through a groupselected from the group consisting of —C(O)NH—, —NR—, —C(O)—, —NHC(O)O—,—NHC(O)NH—, —S—S—, —O—, —(O)CCH₂CH₂C(O)—, and —NHC(O)CH₂CH₂C(O)NH—. 20.The compound of any one of claims 1-19, wherein L is —C(O)NR^(b)—,—NR^(b)—, —C(O)—, —NR^(b)C(O)O—, —NR^(b)C(O)NR^(b)—, —S—S—, —O—,—(O)CCH₂CH₂C(O)—, or —NHC(O)CH₂CH₂C(O)NH—.
 21. The compound of any oneof claims 1-19, wherein L is —NR^(b)—.
 22. The compound of any one ofclaims 1-21, wherein R^(a) is a branched (C₁₀-C₅₀)alkyl or branched(C₁₀-C₅₀)alkenyl, wherein two or more carbon atoms of the branched(C₁₀-C₅₀)alkyl or branched (C₁₀-C₅₀)alkenyl have been replaced with —O—.23. The compound of any one of claims 1-21, wherein R^(a) is a branched(C₁₀-C₅₀)alkyl or branched (C₁₀-C₅₀)alkenyl, wherein two of the branched(C₁₀-C₅₀)alkyl or branched (C₁₀-C₅₀)alkenyl have been replaced with —O—.24. The compound of any one of claims 1-21, wherein R^(a) is a branched(C₁₀-C₅₀)alkyl, wherein two of the branched (C₁₀-C₅₀)alkyl or branched(C₁₀-C₅₀)alkenyl have been replaced with —O—.
 25. The compound of anyone of claims 1-24, wherein each R^(b) is independently H.
 26. Thecompound of any one of claims 1-19, wherein C has the followingstructure:

wherein: R¹ and R² are each independently (C₁₀-C₂₀)alkyl or(C₁₀-C₂₀)alkenyl; M is a direct bond or a divalent (C₁-C₅)alkyl; L isselected from the group consisting of a direct bond, —C(O)O—,—C(O)NR^(b), —NR^(b)—, —C(O)—, —NR^(b)C(O)O—, —NR^(b)C(O)NR^(b)—, —S—S—,—O—, —(O)CCH₂CH₂C(O)—, and —NHC(O)CH₂CH₂C(O)NH—; and each R^(b) isindependently H or (C₁-C₆)alkyl.
 27. The compound of any one of claims1-19, wherein L is selected from the group consisting of —C(O)NR^(b)—,—NR^(b)—, —C(O)—, —NR^(b)C(O)O—, —NR^(b)C(O)NR^(b)—, —S—S—, —O—,—(O)CCH₂CH₂C(O)—, and —NHC(O)CH₂CH₂C(O)NH—.
 28. The compound of any oneof claims 26-27, wherein L is —NR^(b)—.
 29. The compound of any one ofclaims 26-28, wherein M is a direct bond.
 30. The compound of any one ofclaims 26-28, wherein M is a divalent (C₁-C₅)alkyl.
 31. The compound ofany one of claims 26-30, wherein R¹ is selected from the groupconsisting of lauryl (C12), myristyl (C14), palmityl (C16), stearyl(C18) and icosyl (C20).
 32. The compound of any one of claims 26-30,wherein R² is selected from the group consisting of lauryl (C12),myristyl (C14), palmityl (C16), stearyl (C18) and icosyl (C20).
 33. Thecompound of any one of claims 26-30, wherein, wherein R¹ and R² are thesame.
 34. The compound of any one of claims 26-30, wherein, wherein R¹and R² are both lauric (C12).
 35. The compound of any one of claims26-30, wherein, wherein R¹ and R² are both myristyl (C14).
 36. Thecompound of any one of claims 26-30, wherein R¹ and R² are both palmityl(C16).
 37. The compound of any one of claims 26-30, wherein R¹ and R²are both stearyl (C18).
 38. A nucleic acid-lipid particle comprising:one or more nucleic acid molecules; a cationic lipid; a non-cationiclipid; and a compound of formula (I) as described in any one of claims1-37; wherein the one or more nucleic acid molecules are encapsulatedwithin the lipid particle.
 39. The nucleic acid-lipid particle of claim38, wherein said cationic lipid is selected from the group consisting ofN,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and mixturesthereof.
 40. The nucleic acid-lipid particle of claim 38 or 39, whereinsaid non-cationic lipid is a member selected from the group consistingof dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC),palmitoyloleyolphosphatidylglycerol (POPG), cholesterol, and a mixturethereof.
 41. The nucleic acid-lipid particle of claim 38 or 39, whereinsaid non-cationic lipid is an anionic lipid.
 42. The nucleic acid-lipidparticle of claim 38 or 39, wherein said non-cationic lipid is a neutrallipid.
 43. The nucleic acid-lipid particle of any one of claims 38-42,wherein the cationic lipid comprises from about 5% to about 90% of thetotal lipid present in said particle.
 44. The nucleic acid-lipidparticle of any one of claims 38-42, wherein the cationic lipidcomprises from about 15% to about 90% of the total lipid present in saidparticle.
 45. The nucleic acid-lipid particle of any one of claims38-42, wherein the cationic lipid comprises from about 25% to about 90%of the total lipid present in said particle.
 46. The nucleic acid-lipidparticle of any one of claims 38-42, wherein the cationic lipidcomprises from about 5% to about 80% of the total lipid present in saidparticle.
 47. The nucleic acid-lipid particle of any one of claims38-42, wherein the cationic lipid comprises from about 15% to about 70%of the total lipid present in said particle.
 48. The nucleic acid-lipidparticle of any one of claims 38-42, wherein the cationic lipidcomprises from about 25% to about 60% of the total lipid present in saidparticle.
 49. The nucleic acid-lipid particle of any one of claims38-42, wherein the cationic lipid comprises from about 40% to about 60%of the total lipid present in said particle.
 50. The nucleic acid-lipidparticle of any one of claims 38-49, wherein the non-cationic lipid isDSPC.
 51. The nucleic acid-lipid particle of any one of claims 38-50,wherein the non-cationic lipid comprises cholesterol.
 52. The nucleicacid-lipid particle of claim 58, wherein the cholesterol comprises fromabout 10% to about 60% of the total lipid present in said particle. 53.The nucleic acid-lipid particle of claim 58, wherein the cholesterolcomprises from about 20% to about 45% of the total lipid present in saidparticle.
 54. The nucleic acid-lipid particle of any one of claims38-53, wherein the nucleic acid is DNA.
 55. The nucleic acid-lipidparticle of any one of claims 38-53, wherein the nucleic acid is RNA.56. The nucleic acid-lipid particle of any one of claims 38-53, whereinthe nucleic acid is selected from the group consisting of smallinterfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA(shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA,tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, guide RNA forgene editing systems, DNA, plasmids, antisense oligonucleotides, andcombinations thereof.
 57. The nucleic acid-lipid particle of any one ofclaims 38-53, wherein the nucleic acid is a ribozyme.
 58. The nucleicacid-lipid particle of any one of claims 38-53, wherein the nucleic acidis a small interfering RNA (siRNA).
 59. The nucleic acid-lipid particleof any one of claims 38-53, wherein the nucleic acid is mRNA, andwherein the mRNA encodes a therapeutic product of interest.
 60. Thenucleic acid-lipid particle of claim 59, wherein said therapeuticproduct of interest is a peptide or protein.
 61. The nucleic acid-lipidparticle of claim 59, wherein said therapeutic product of interest is avaccine antigen.
 62. The nucleic acid-lipid particle of any one ofclaims 38-61, that is not substantially degraded after exposure of saidparticle to a nuclease at 37° C. for 20 minutes.
 63. The nucleicacid-lipid particle of any one of claims 38-61, that is notsubstantially degraded after incubation of said particle in serum at 37°C. for 30 minutes.
 64. The nucleic acid-lipid particle of any one ofclaims 38-61, wherein the one or more nucleic acid molecules are fullyencapsulated in said nucleic acid-lipid particle.
 65. The nucleicacid-lipid particle of any one of claims 38-61, wherein the nucleicacid-lipid particle has a lipid:nucleic acid mass ratio of from about 5to about
 30. 66. The nucleic acid-lipid particle of claim 65, whereinthe nucleic acid-lipid particle has a lipid:nucleic acid mass ratio offrom about 5 to about
 15. 67. The nucleic acid-lipid particle of claim64, wherein the nucleic acid is mRNA and wherein the nucleic acid-lipidparticle has a lipid:nucleic acid mass ratio of from about 15 to about25.
 68. A pharmaceutical composition comprising a nucleic acid-lipidparticle of any one of claims 38-67, and a pharmaceutically acceptablecarrier.
 69. A composition comprising about 1.5% of total lipid of thecompound:

wherein n is selected so that the resulting polymer chain has amolecular weight of about 2000 daltons; about 50.0% of total lipid ofthe compound:

about 38.5% of total lipid of cholesterol; and about 10.0% of totallipid of DSPC.
 70. A composition comprising about 2.0% of total lipid ofthe compound:

wherein n is selected so that the resulting polymer chain has amolecular weight of about 2000 daltons; about 40.0% of total lipid ofthe compound:

about 48.5% of total lipid of cholesterol; and about 10.0% of totallipid of DSPC.
 71. A composition comprising about 1.6% of total lipid ofthe compound:

wherein n is selected so that the resulting polymer chain has amolecular weight of about 2000 daltons; about 54.9% of total lipid ofthe compound:

about 32.8% of total lipid of cholesterol; and about 10.0% of totallipid of DSPC.
 72. The composition of any one of claims 69-71 furthercomprising one or more nucleic acid molecules.
 73. The composition ofclaim 72, wherein the nucleic acid is mRNA.
 74. A method of introducinga nucleic acid into a cell, comprising contacting said cell with anucleic acid-lipid particle of any one of claims 38-67 or a compositionof any one of claims 68-73.
 75. The method of claim 74, wherein the cellis in a mammal.
 76. A method for the in vivo delivery of a nucleic acid,the method comprising: administering to a mammalian subject a nucleicacid-lipid particle of any one of claims 45-74 or a composition of anyone of claims 68-73.
 77. The method of claim 76, wherein theadministration is selected from the group consisting of oral,intranasal, intravenous, intraperitoneal, intramuscular,intra-articular, intralesional, intratracheal, subcutaneous, andintradermal.
 78. The method of claim 77, wherein the administration isintramuscular.
 79. A nucleic acid-lipid particle of any one of claims38-67 or a composition of any one of claims 68-73 for use in the in vivodelivery of a nucleic acid to a mammal.
 80. The use of a nucleicacid-lipid particle of any one of claims 38-67 or a composition of anyone of claims 68-73 to prepare a medicament for the in vivo delivery ofa nucleic acid to a mammal.
 81. A method for treating a disease ordisorder in a mammalian subject in need thereof, the method comprising:administering to the mammalian subject a therapeutically effectiveamount of nucleic acid-lipid particle of any one of claims 38-67 or acomposition of any one of claims 68-73.
 82. The method of claim 81,wherein the disease or disorder is selected from the group consisting ofa monogenic disorder, a viral infection, a liver disease or disorder,and cancer.
 83. A nucleic acid-lipid particle of any one of claims 38-67or a composition of any one of claims 75-80 for use in treating adisease or disorder in a mammal.
 84. The use of a nucleic acid-lipidparticle of any one of claims 38-67 or a composition of any one ofclaims 68-73 to prepare a medicament for treating a disease or disorderin a mammal.